Methods and compositions for immunization against virus

ABSTRACT

Immunogenic compositions comprising partially glycosylated viral glycoproteins for use as vaccines against viruses are provided. Vaccines formulated using mono-, di-, or tri-glycosylated viral surface glycoproteins and polypeptides provide potent and broad protection against viruses, even across strains. Pharmaceutical compositions comprising monoglycosylated hemagglutinin polypeptides and vaccines generated therefrom and methods of their use for prophylaxis or treatment of viral infections are disclosed. Methods and compositions are disclosed for influenza virus HA, NA and M2, RSV proteins F, G and SH, Dengue virus glycoproteins M or E, hepatitis C virus glycoprotein E1 or E2 and HIV glycoproteins gp120 and gp41.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/182,296, filed Feb. 18, 2014, which is a division of U.S.application Ser. No. 12/748,265, filed Mar. 26, 2010 which claimspriority of U.S. provisional patent application Ser. No. 61/164,385,titled “METHODS AND COMPOSITIONS FOR IMMUNIZATION AGAINST INFLUENZA”filed Mar. 27, 2009, U.S. provisional patent application Ser. No.61/164,387, titled “METHODS AND COMPOSITIONS FOR IMMUNIZATION AGAINSTHUMAN IMMUNODEFICIENCY VIRUS” filed Mar. 28, 2009, U.S. provisionalpatent application Ser. No. 61/164,388, titled “METHODS AND COMPOSITIONSFOR IMMUNIZATION AGAINST FLAVIVIRUS” filed Mar. 28, 2009, U.S.provisional patent application Ser. No. 61/164,389, titled “METHODS FORMANUFACTURING VACCINES AGAINST VIRAL INFECTION” filed Mar. 28, 2009,U.S. provisional patent application Ser. No. 61/313,676, titled “METHODSAND COMPOSITIONS FOR IMMUNIZATION AGAINST INFLUENZA” filed Mar. 12,2010, the contents of each of which are incorporated herein in theirentirety by reference.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing. A computer readable copyof the Sequence Listing was submitted by EFS-Web on Jun. 4, 2019 as anASCII file created on Jun. 4, 2019, named A098870064US07-SEQ-JAV, whichis 62,828 bytes in size. The information contained in the SequenceListing is herein incorporated by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

The invention generally relates to partially glycosylated viralpolypeptides that are useful for generating potent, broadly-reactiveimmunogenic compositions effective against the virus. In particular, theinvention relates to vaccines generated using monoglycosylated influenzavirus hemagglutinin (HA) peptide, the vaccines exhibiting potentactivity against influenza viruses.

The invention relates to pharmaceutical compositions comprising theglycoproteins and vaccines generated therefrom, and to methods of usingthe deglycosylated HA polypeptides for prophylaxis and treatment ofinfluenza virus infections

BACKGROUND OF THE INVENTION

In eukaryotes, sugar residues are commonly linked to four differentamino acid residues. These amino acid residues are classified asO-linked (serine, threonine, and hydroxylysine) and N-linked(asparagine). The O-linked sugars are synthesized in the Golgi or roughEndoplasmic Reticulum (ER) from nucleotide sugars. The N-linked sugarsare synthesized from a common precursor, and subsequently processed. Itis known that addition of N-linked carbohydrate chains is important forstabilization of folding, prevention of degradation in the endoplasmicreticulum, oligomerization, biological activity, and transport ofglycoproteins. The addition of N-linked oligosaccharides to specific Asnresidues plays an important role in regulating the activity, stabilityor antigenicity of mature proteins of viruses (Opdenakker G. et al FASEBJournal 7, 1330-1337 1993). It has also been suggested that N-linkedglycosylation is required for folding, transport, cell surfaceexpression, secretion of glycoproteins (Helenius, A., Molecular Biologyof the Cell 5, 253-265 1994), protection from proteolytic degradationand enhancement of glycoprotein solubility (Doms et al., Virology 193,545-562 1993). Viral surface glycoproteins are not only required forcorrect protein folding, but also provide protection againstneutralizing antibodies as a “glycan shield.” As a result, stronghost-specific selection is frequently associated with codon positions ofpotential N-linked glycosylation. Consequently N-linked glycosylationsites tend to be conserved across strains and clades.

There are three main types of influenza virus: A, B and C. Type Astrains of influenza virus can cause severe illness and are the onlytype to have caused human pandemics. The H5N1 strain is a type Ainfluenza virus. Type B strains cause sporadic human cases andsmall-scale outbreaks. Type C strains only rarely cause human infectionand have not caused large outbreaks. Of the influenza A viruses, onlysubtypes HE H2 and H3 have been transmitted easily between humans.

Outbreaks of influenza A virus continue to cause widespread morbidityand mortality worldwide. In the United States alone, an estimated 5 to20% of the population is infected by influenza A virus annually, causingapproximately 200,000 hospitalizations and 36,000 deaths. Theestablishment of comprehensive vaccination policies has been aneffective measure to limit influenza morbidity. However, the frequentgenetic drifting of the virus requires yearly reformulation of thevaccine, potentially leading to a mismatch between the viral strainpresent in the vaccine and that circulating. Thus, antiviral therapiesagainst influenza virus are important tools to limit both diseaseseverity as well as transmission.

The highly pathogenic H5N1 influenza viruses have caused outbreaks inpoultry and wild birds since 2003 (Li K S et al. (2004) Nature430:209-213). As of February 2010, these viruses have infected not onlyavian species but also over 478 humans, of which 286 cases proved to befatal(www.who.int/csr/disease/avian_influenza/country/cases_table_2010_02_17/en/index.html).The highly pathogenic H5N1 and the 2009 swine-origin influenza A (H1N1)viruses have caused global outbreaks and raised a great concern thatfurther changes in the viruses may occur to bring about a deadlypandemic (Garten R J, et al. (2009) Science 325:197-201, Neumann G, etal. (2009) Nature 459:931-939). There is great concern that an influenzavirus would acquire the ability to spread efficiently between humans,thereby becoming a pandemic threat. An influenza vaccine must,therefore, be an integral part of any pandemic preparedness plan.

Influenza viruses are segmented negative-strand RNA viruses and belongto the Orthomyxoviridae family. Influenza A virus consists of 9structural proteins and codes additionally for one nonstructural NS1protein with regulatory functions. The non-structural NS1 protein issynthesized in large quantities during the reproduction cycle and islocalized in the cytosol and nucleus of the infected cells. Thesegmented nature of the viral genome allows the mechanism of geneticreassortment (exchange of genome segments) to take place during mixedinfection of a cell with different viral strains. The influenza A virusmay be further classified into various subtypes depending on thedifferent hemagglutinin (HA) and neuraminidase (NA) viral proteinsdisplayed on their surface. Influenza A virus subtypes are identified bytwo viral surface glycoproteins, hemagglutinin (HA or H) andneuraminidase (NA or N). Each influenza virus subtype is identified byits combination of H and N proteins. There are 16 known HA subtypes and9 known NA subtypes. Influenza type A viruses can infect people, birds,pigs, horses, and other animals, but wild birds are the natural hostsfor these viruses. Only some influenza A subtypes (i.e., H1N1, H1N2, andH3N2) are currently in circulation among people, but all combinations ofthe 16H and 9 NA subtypes have been identified in avian species,especially in wild waterfowl and shorebirds. In addition, there isincreasing evidence that H5 and H7 influenza viruses can also causehuman illness.

The HA of influenza A virus comprises two structurally distinct regions,namely, a globular head region and a stem region. The globular headregion contains a receptor binding site which is responsible for virusattachment to a target cell and participates in the hemagglutinationactivity of HA. The stem region contains a fusion peptide which isnecessary for membrane fusion between the viral envelope and anendosomal membrane of the cell and thus relates to fusion activity(Wiley et al., Ann Rev. Biochem., 56:365-394 (1987)).

Important contributions to the understanding of influenza infectionshave come from the studies on hemagglutinin (HA), a viral coatglycoprotein that binds to specific sialylated glycan receptors in therespiratory tract, allowing the virus to enter the cell (Kuiken T, etal. (2006) Science 312:394-397; Maines T R, et al. (2009) Science325:484-487; Skehel J J, Wiley D C (2000) Ann Rev Biochem 69:531-569;van Riel D, et al. (2006) Science 312:399-399). To cross the speciesbarrier and infect the human population, avian HA must change itsreceptor-binding preference from a terminally sialylated glycan thatcontains α2,3 (avian)-linked to α2,6 (human)-linked sialic acid motifs(Connor R J, et al. (1994) Virology 205:17-23), and this switch couldoccur through only two mutations, as in the 1918 pandemic (Tumpey T M,et al. (2007) Science 315:655-659). Therefore, understanding the factorsthat affect influenza binding to glycan receptors is critical fordeveloping methods to control any future crossover influenza strainsthat have pandemic potential.

To address the need for making a candidate influenza vaccine that couldinduce potent neutralizing antibodies against divergent strains of H5N1influenza viruses a consensus H5N1 hemagglutinin (HA) sequence basedvaccine elicited antibodies that neutralized a panel of virions thathave been pseudotyped with the HA from various H5N1 clades. (Chen M W,et al. (2008) Proc Natl Acad Sci USA 105:13538-13543).

HA is a homotrimeric transmembrane protein with an ectodomain composedof a globular head and a stem region (Kuiken T, et al. (2006) Science312:394-397). Both regions carry N-linked oligosaccharides (Keil W, etal. (1985) EMBO J 4:2711-2720), which affect the functional propertiesof HA (Chen Z Y, et al. (2008) Vaccine 26:361-371; Ohuchi R, et al.(1997) J Virol 71:3719-3725). Among different subtypes of influenza Aviruses, there is extensive variation in the glycosylation sites of thehead region, whereas the stem oligosaccharides are more conserved andrequired for fusion activity (Ohuchi R, et al. (1997) J Virol71:3719-3725). Glycans near antigenic peptide epitopes interfere withantibody recognition (Skehel J J, et al. (1984) Proc Natl Acad Sci USA81:1779-1783), and glycans near the proteolytic activation site of HAmodulate cleavage and influence the infectivity of influenza virus(Deshpande K L, et al. (1987) Proc Natl Acad Sci USA 84:36-40).Mutational deletion of HA glycosylation sites can affect viral receptorbinding (Gunther I, et al. (1993) Virus Res 27:147-160).

Changes in the peptide sequence at or near glycosylation sites may alterHA's 3D structure, and thus receptor-binding specificity and affinity.Indeed, HAs from different H5N1 subtypes have different glycan-bindingpatterns (Stevens J, et al. (2008) J Mol Biol 381:1382-1394).Mutagenesis of glycosylation sites on H1 and H3 has been studied in thewhole-viral system (Chandrasekaran A, et al. (2008) Nat Biotechnol26:107-113; Deom C M, et al. (1986) Proc Natl Acad Sci USA83:3771-3775). However, it is not known how changes in glycosylationaffect receptor-binding specificity and affinity, especially with regardto the most pathogenic H5N1 HA.

Flu vaccines, when made, have to be changed every year as the lesshighly glycosylated or non-glycosylated regions of hemagglutinincontinue to mutate to escape from the host immune system.

The goal of vaccine design against heterogeneous pathogens is toidentify and design effective and broadly protective antigens. In thecase of influenza, considerable historical efforts have gone into theempirical testing of conserved linear sequences and regions with littlesuccess. A plausible reason for these failures is a lack of knowledgethat focused responses against antigenic test articles are actual bonafide productive sites for neutralization of an antigen on the pathogenin the setting of an actual infection.

SUMMARY OF THE INVENTION

Specifically, there is a need for cross neutralizing monoclonalantibodies that can be used in the design and validation of vaccineproduction processes that maintain or enhance the quality andantigenicity of cross neutralizing epitopes in current and futuremanufactured vaccines. Assuming that antibody binding to vaccine isreflective of structural integrity and antigenic potential, one wouldassess binding of cross neutralizing antibodies, such as deglycosylatedHA polypeptides to such vaccine process derivatives to quantitativelyassess their cross neutralizing potential. To maximize the responsestoward these universal epitopes one would create derivatives to increaseimmunogenicity towards these universal epitopes.

According to the invention, a vaccine using these principles isdisclosed. The antigen is generated by partially removing sugars fromthe viral glycoprotein to expose the glycosylation sites (which arehighly conserved and do not mutate or do not mutate aggressively) and atthe same time retain adequate sugars to preserve the tertiary structureof the glycoprotein. The partially glycosylated viral glycoproteins aregenerated by partially deglycosylating the glycoproteins such that aparticular glycosylation site retains one, two or three sugar units. Insome aspects the partially glycosylated glycoprotein can be generated byproviding a protein or polypeptide unglycosylated at one or moreparticular glycosylation sites and conjugating a mono-, di- ortri-saccharide to the glycosylation sites.

A vaccine is disclosed comprising at least one partially glycosylatedHA, NA or M2 glycoprotein and a pharmaceutically acceptable carrier. Insome implementations, the partially glycosylated HA glycoprotein isselected from the group consisting of partially glycosylated influenzavirus HI, H3, and H5. In some implementations, the partiallyglycosylated HA glycoprotein is glycosylated at asparagine residues atone or more of positions 39, 127, 170, 181, 302, 495 and 500 of H5 HA.In some implementations, the asparagine residue is at position 177.

A method is disclosed comprising administering to a subject susceptibleof influenza a vaccine comprising at least one deglycosylated HAglycoprotein and a pharmaceutically acceptable carrier. In someimplementations, the deglycosylated HA glycoprotein is selected from thegroup consisting of HI, H3, and H5.

In some implementations the deglycosylation leaves a monoglycosylation(one sugar remaining) at one or more glycosylation site on theglycoprotein. In some implementations the deglycosylation leaves adiglycosylation (2 sugars remaining) at at least one glycosylation siteon the glycoprotein. In some implementations the deglycosylation leavesa triglycosylation (3 sugars remaining) at one or more glycosylationsite on the glycoprotein. In some implementations the deglycosylationleaves at least one of a monoglycosylation, a diglycosylation and atriglycosylation at at least one glycosylation site on the glycoprotein.

The invention relates to an immunogenic composition for raising animmune response to a pathogen of viral, bacterial, fungal or otherorigin, the composition comprising: an antigen glycoprotein from thepathogen of viral, bacterial, fungal or other origin, wherein theglycoprotein is partially glycosylated.

In some aspects, the pathogen is a virus and the partially glycosylatedantigen is a virus, a virus-like particle, a viral peptide, a protein, apolypeptide, or a fragment thereof derived from the virus, or a fusionprotein partially comprising a virus protein sequence.

The virus is selected from influenza virus, respiratory syncytial virus(RSV), chlamydia, adenovirdiae, mastadenovirus, aviadenovirus,herpesviridae, herpes simplex virus 1, herpes simplex virus 2, herpessimplex virus 5, herpes simplex virus 6, leviviridae, levivirus,enterobacteria phase MS2, allolevirus, poxviridae, chordopoxvirinae,parapoxvirus, avipoxvirus, capripoxvirus, leporiipoxvirus, suipoxvirus,molluscipoxvirus, entomopoxvirinae, papovaviridae, polyomavirus,papillomavirus, paramyxoviridae, paramyxovirus, parainfluenza virus 1,mobillivirus, measles virus, rubulavirus, mumps virus, pneumonovirinae,pneumovirus, metapneumovirus, avian pneumovirus, human metapneumovirus,picornaviridae, enterovirus, rhinovirus, hepatovirus, human hepatitis Avirus, cardiovirus, andapthovirus, reoviridae, orthoreovirus, orbivirus,rotavirus, cypovirus, fijivirus, phytoreovirus, oryzavirus,retroviridae, mammalian type B retroviruses, mammalian type Cretroviruses, avian type C retroviruses, type D retrovirus group,BLV-HTLV retroviruses, lentivirus, human immunodeficiency virus 1, humanimmunodeficiency virus 2, HTLV-I and -II viruses, SARS coronavirus,herpes simplex virus, Epstein Barr virus, cytomegalovirus, hepatitisvirus (HCV, HAV, HBV, HDV, HEV), Toxoplasma gondii virus, treponemapallidium virus, human T-lymphotrophic virus, encephalitis virus, WestNile virus, Dengue virus, Varicella Zoster Virus, rubeola, mumps,rubella, spumavirus, flaviviridae, hepatitis C virus, hepadnaviridae,hepatitis B virus, togaviridae, alphavirus sindbis virus, rubivirus,rubella virus, rhabdoviridae, vesiculovirus, lyssavirus, ephemerovirus,cytorhabdovirus, necleorhabdovirus, arenaviridae, arenavirus,lymphocytic choriomeningitis virus, Ippy virus, lassa virus,coronaviridae, coronavirus and torovirus.

The viral peptide, protein, polypeptide, or a fragment thereof isselected from influenza virus neuraminidase, influenza virushemagglutinin, human respiratory syncytial virus (RSV)-viral proteins,RSV F glycoprotein, RSV G glycoprotein, herpes simplex virus (HSV) viralproteins, herpes simplex virus glycoproteins gB, gC, gD, and gE,chlamydia MOMP and PorB antigens, core protein, matrix protein or otherprotein of Dengue virus, measles virus hemagglutinin, herpes simplexvirus type 2 glycoprotein gB, poliovirus 1 VP1, envelope glycoproteinsof HIV 1, hepatitis B surface antigen, diptheria toxin, streptococcus24M epitope, gonococcal pilin, pseudorabies virus g50 (gpD),pseudorabies virus II (gpB), pseudorabies virus III (gpC), pseudorabiesvirus glycoprotein H, pseudorabies virus glycoprotein E, transmissiblegastroenteritis glycoprotein 195, transmissible gastroenteritis matrixprotein, swine rotavirus glycoprotein 38, swine parvovirus capsidprotein, Serpulinahydodysenteriae protective antigen, bovine viraldiarrhea glycoprotein 55, Newcastle disease virushemagglutinin-neuraminidase, swine flu hemagglutinin, swine fluneuraminidase, foot and mouth disease virus, hog colera virus, swineinfluenza virus, African swine fever virus, Mycoplasma liyopneutiioniae,infectious bovine rhinotracheitis virus, infectious bovinerhinotracheitis virus glycoprotein E, glycoprotein G, infectiouslaryngotracheitis virus, infectious laryngotracheitis virus glycoproteinG or glycoprotein I, a glycoprotein of La Crosse virus, neonatal calfdiarrhoea virus, Venezuelan equine encephalomyelitis virus, punta torovirus, murine leukemia virus, mouse mammary tumor virus, hepatitis Bvirus core protein and hepatitis B virus surface antigen or a fragmentor derivative thereof, antigen of equine influenza virus or equineherpes virus, including equine influenza virus type A/Alaska 91neuraminidase, equine influenza virus typeA/Miami 63 neuraminidase,equine influenza virus type A/Kentucky 81 neuraminidase equine herpesvirus type 1 glycoprotein B, and equine herpes virus type 1 glycoproteinD, antigen of bovine respiratory syncytial virus or bovine parainfluenzavirus, bovine respiratory syncytial virus attachment protein (BRSV G),bovine respiratory syncytial virus fusion protein (BRSV F), bovinerespiratory syncytial virus nucleocapsid protein (BRSVN), bovineparainfluenza virus type 3 fusion protein, bovine parainfluenza virustype 3 hemagglutinin neuraminidase, bovine viral diarrhoea virusglycoprotein 48 and glycoprotein 53, glycoprotein E of Dengue virus andglycoprotein E1E2 of human hepatitis C virus.

In some aspects, the deglycosylated viral antigen is a mono-, di-, ortri-glycosylated influenza virus hemagglutinin. In some embodiments, thedeglycosylated viral antigen is a mono-glycosylated hemagglutininselected from the group consisting of influenza virus HI, 113, and H5.In some embodiments, the mono-glycosylated influenza virus hemagglutinincomprises an N-glycosylation site comprising an amino acid sequence ofasparagine-X_(aa)-serine and asparagine-X_(aa)-threonine, where X_(aa)is any amino acid except proline. In some aspects, the monoglycosylatedhemagglutinin comprising a single GlcNAc sugar at a glycosylation sitedisplays relaxed specificity but enhanced affinity towards HA-receptorbinding.

In some embodiments, the deglycosylated viral antigen is an influenzavirus hemagglutinin di- or tri-glycosylated with N-acetylglucosamine(GlcNAc) and/or mannose. In some aspects, the deglycosylated viralantigen is a mono-glycosylated influenza virus hemagglutininglycosylated with N-acetylglucosamine (GlcNAc).

In some aspects, the mono-glycosylated influenza virus hemagglutinincomprises a polypeptide comprising a consensus H5 HA sequence (SEQ IDNO: 4). In some embodiments, the mono-glycosylated consensus H5 HAsequence (SEQ ID NO: 4) is glycosylated at asparagine residues at one ormore of positions 39, 170, 181, 302 and 495. In other aspects, themono-glycosylated influenza virus hemagglutinin comprises a H1polypeptide sequence selected from the group consisting of a NIBRG-121(Pandemic 2009 A(H1N1) vaccine strain) sequence (SEQ ID NO: 6), aconsensus H1-A (SEQ ID NO: 8) and a consensus H1-C(SEQ ID NO: 10)sequence. In some embodiments, the HA sequence is modified to enableexpression in a suitable eukaryotic cell.

In one embodiment, the mono-glycosylated influenza virus hemagglutinincomprises a seasonal H1 (Brisbane) polypeptide.

The invention also relates to a vaccine comprising an immunogenicpolypeptide comprising a viral glycoprotein deglycosylated to a state ofmono-, di-, or tri-glycosylation and optionally, an adjuvant, whereinthe vaccine is capable of eliciting an immune response against arespiratory virus. In some embodiments, the respiratory virus is aninfluenza virus and the viral glycoprotein is hemagglutinin (HA).

In some aspects, the influenza virus is selected from the groupconsisting of an avian influenza virus and a seasonal influenza virus.In some embodiments, the avian influenza virus is H5N1. In someembodiments, the influenza virus is influenza A virus.

In one aspect, the virus is respiratory syncytial virus (RSV), and thepartially glycosylated viral antigen is a mono-, di-, ortri-glycosylated RSV F (fusion), G (attachment) of SH (smallhydrophobic) glycoprotein, or immunogenic fragments thereof. In someembodiments, the mono-glycosylated RSV G protein sequence (SEQ ID NO:12) is partially glycosylated at asparagine residues at one or morepotential N-glycosylation sites indicated in Table 6.

In one aspect, the virus is a flavivirus, and the partially glycosylatedviral antigen is a mono-, di-, or tri-glycosylated Dengue virus envelopeglycoprotein M, glycoprotein E, or immunogenic fragments thereof. Insome embodiments, the mono-glycosylated Dengue virus envelopeglycoprotein E (SEQ ID NO: 13) is partially glycosylated at asparagineresidues at one or more N-glycosylation sites N67 and N153 indicated inTable 7.

In one aspect, the virus is a hepatitis C virus, and the partiallyglycosylated viral antigen is a mono-, di-, or tri-glycosylatedhepatitis C envelope glycoprotein E1, glycoprotein E2, or immunogenicfragments thereof. In some embodiments, the mono-glycosylated hepatitisC envelope glycoprotein E1 (SEQ ID NO: 14) is partially glycosylated atasparagine residues at one or more N-glycosylation sites N196, N209,N234, N305, AND N325 indicated in Table 8.

In one aspect, wherein the virus is a human immunodeficiency virus(HIV), and the partially glycosylated viral antigen is a mono-, di-, ortri-glycosylated HIV envelope glycoprotein gp120, transmembraneglycoprotein gp41, or immunogenic fragments thereof. In someembodiments,

the mono-glycosylated HIV envelope glycoprotein gp120 (SEQ ID NO: 15) ispartially glycosylated at asparagine residues at one or more potentialN-glycosylation sites indicated in Table 9.

The invention also relates to a vaccine composition comprising: aninfluenza HA polypeptide, wherein the influenza HA polypeptide isdeglycosylated to a state of monoglycosylation; and a pharmaceuticallyacceptable carrier, wherein upon introduction of the mono-glycosylatedHA polypeptide into a subject, the polypeptide induces the subject toproduce antibodies that bind to influenza virus.

In some aspects, introduction of the mono-glycosylated HA polypeptideinto the subject, induces the subject to produce antibodies thatneutralize both seasonal and avian influenza virus.

In one embodiment, the mono-glycosylated influenza virus hemagglutinincomprises a seasonal H1 (Brisbane) HA polypeptide and upon introductionof the mono-glycosylated HA polypeptide into a subject, the H1(Brisbane) HA polypeptide induces the subject to produce antibodies thatneutralize NIBRG-121 (Pandemic 2009 A(H1N1) vaccine strain) influenzavirus.

In another embodiment, the mono-glycosylated influenza virushemagglutinin comprises a NIBRG-121 (Pandemic 2009 A(H1N1) vaccinestrain) polypeptide and upon introduction of the mono-glycosylated HApolypeptide into a subject, the NIBRG-121 (Pandemic 2009 A(H1N1) vaccinestrain) polypeptide induces the subject to produce antibodies thatneutralize seasonal H1 (Brisbane) HA influenza virus.

In some aspects, the vaccine further comprises an adjuvant, which can beselected from aluminum hydroxide, aluminum phosphate, both aluminumhydroxide and aluminum phosphate, incomplete Freund's adjuvant (IFA),squalene, squalane, alum, and MF59.

The invention relates to methods for immunizing a mammal against a viralrespiratory infection, the method comprising: administering to themammal susceptible to infection by the respiratory virus a vaccinecomprising an immunogenic polypeptide comprising a viral glycoproteindeglycosylated to a state of mono-, di-, or tri-glycosylation, whereinthe vaccine is capable of eliciting an immune response against therespiratory virus. In some embodiments, the respiratory virus is aninfluenza virus and the viral glycoprotein is hemagglutinin (HA). Thevaccine may be administered through parenteral administration,inhalation means, intranasally, and sometimes prophylactically.

In some aspects, the vaccine elicits immune response against influenzavirus strains that are different from the influenza virus strain fromwhich the deglycosylated viral glycoprotein is selected. In someembodiments, the deglycosylated viral glycoprotein is amono-glycosylated influenza hemagglutinin (HA).

The present invention provides vaccines effective against influenza Avirus. In one embodiment, the vaccine comprises a peptide or polypeptidefunctionally mimicking a neutralization epitope of a molecule describedherein. In another embodiment, the vaccine is effective against a viralantigen comprises a peptide or polypeptide functionally mimicking aneutralization epitope of a molecule described herein. In oneembodiment, the viral antigen is from an influenza virus or an HIV-1 orHIV-2 virus, or a flavivirus, such as Dengue virus or hepatitis C virus.

In another embodiment, the vaccine is a vaccine effective againstinfluenza A virus, comprising a peptide or polypeptide functionallymimicking a neutralization epitope of a molecule described herein. Inone embodiment, the molecule is an antibody. In another embodiment, theantibody binds an HA antigen. In one other embodiment, the HA antigen isan H5 subtype. In one other embodiment, the HA antigen is an H1 subtype.In one other embodiment, the antigen is displayed on the surface ofinfluenza A virus. In one other embodiment, the peptide or polypeptidecomprises antigenic determinants that raise neutralizing antibodies.

These and other aspects will become apparent from the followingdescription of the preferred embodiment taken in conjunction with thefollowing drawings, although variations and modifications therein may beaffected without departing from the spirit and scope of the novelconcepts of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, the inventions of which can be better understood byreference to one or more of these drawings in combination with thedetailed description of specific embodiments presented herein. Thepatent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1C show schematic overviews and circular dichroism spectra ofHAs with different glycosylations. (FIG. 1A) Four variants of HAproteins with different glycosylations: HA_(fg), HA (a consensussequence (Chen M W, et al. (2008) Proc Natl Acad Sci USA105:13538-13543) expressed in HEK293E cells with the typical complextype N-glycans); HA_(ds), NA-treated HA resulting in removal of sialicacids from HA_(fg); HA expressed in GnTI-HEK293S cells with thehigh-mannose-type N-glycans; and HA_(mg), Endo H-treated HA with GlcNAconly at its N-glycosylation sites. (FIG. 1B) Structure representation ofHA_(fg), HA_(ds), HA_(hm), and HA_(mg) with different N-glycans attachedat their N-glycosylation sites. The protein structures are created withProtein Data Bank ID code 2FK0 (Viet04 HA), colored in gray, and theN-linked glycans are displayed in green. All N-glycans are modeled byGlyProt (Bohne-Lang A, et al. (2005) Nucleic Acids Res 33:W214-W219),and the graphics are generated by PyMOL (www.pymol.org). (FIG. 1C)Circular dichroism spectra of HA_(fg), HA_(ds), HA_(hm), and HA_(mg)demonstrate that the secondary structures of the four HA proteins withdifferent glycosylations are similar.

FIGS. 2A-2B show glycan microarray analysis of HA with differentglycosylations. (FIG. 2A) Glycan microarray profiling of HA variantsHA_(fg), HA_(ds), HA_(hm), and HA_(mg) are shown. The related linkagesof glycans were grouped by color, predominantly 17 α2,3 sialosides(yellow) or 7 α2,6 sialosides (blue). The structures of glycans on thearray are indicated in FIG. 2B. Association constants of HA variantsHA_(fg), HA_(as), HA_(hm), and HA_(mg) are shown with values ofK_(A,surf) of HA variants in response to α2,3 sialosides 1-15.

FIG. 3 shows Sequence alignment analysis of Brisbane H1, California H1,H3 and H5 of recent HAs H1, H3, and H5 since 2000. Seasonal flu HA isfrom A/Brisbane/59/2007. Pandemic flu HA is from A/California/07/2009.HA from H1N1 A/Brisbane/59/2007 (SEQ ID NO: 16). HA from H1N1A/California/07/2009 (SEQ ID NO: 17). HA from H5N1 A/Vietnam/1203/2004(SEQ ID NO: 18). HA from H3N2 A/Brisbane/10/2007 (SEQ ID NO: 19). H5 isfrom A/Vietnam/1203/2004. H3 is from A/Brisbane/10/2007. TheN-glycosylation sites on H5 HA are shown in a red box. The comparisonreveals similarity between H1 and H5 HAs in their N-glycosylationpositions, whereas H3 has less-conserved glycosylation positions anddiffers from H1 and H5.

FIGS. 4A-4F show the binding energy contributions from sugars ormodifications of HA glycan interactions in response to HAs withdifferent glycosylations. These values were obtained by subtraction ofΔG values of the indicated reference glycan (highlighted in red boxes;see Table S3). (FIG. 4A) Glycans 2, 3, 6, and 8-10 possess the samebackbone of the disaccharide glycan 1 but only differ in the third sugarfrom the nonreducing end. The values of ΔG are calculated to demonstratethe binding energy difference by changing the third sugars. (FIG. 4B)Glycans 10-12 and 15 possess the same backbone of the disaccharideglycan 8 but differ either by elongating the sugar structure linearly orby adding a branched sugar. (FIG. 4C) Glycans 4 and 5 possess the samebackbone of the trisaccharide glycan 3 but differ either by the branchedfucose or the sulfate group on the third position from the nonreducingend. (FIG. 4D) Glycans 6 and 7 differ in the sulfate group on the thirdposition from the nonreducing end of glycan 7. (FIG. 4E) Glycans 13 and14 are α2,3 biantennary sialosides but differ in the change of theinternal sugar. (FIG. 4F) Glycans 16 and 17 and α2,6 sialosides (nos.21-27) show little or no binding to HA.

FIGS. 5A-5D show a comparison of HA_(fg) and HA_(mg) as vaccine. (FIG.5A) The bindings between antisera from HA_(fg) and HA_(mg). and variousHAs are analyzed by using ELISA. In comparison with HA_(fg) antiserum,HA_(mg) antiserum shows better binding to HS (Vietnam 1194/2004 andCHAS). In addition, the HA_(mg) antiserum also binds to H1 (California0712009 and WSN). (FIG. 5B) Microneutralization of H5N1 (NIBRG-14) viruswith HA_(fg) and HA_(mg) antisera. In comparison with HA_(fg) antiserum,HA_(mg) antiserum shows better neutralizing activity against influenzavirus infection to MDCK cells (P<0.0001). (FIG. 5C) Vaccine protectionagainst lethal-dose challenge of H5N1 virus. BALB/c mice were immunizedwith two injections of the HA protein vaccine HA_(fg), HA_(mg). andcontrol PBS. The immunized mice were intranasally challenged with alethal dose of H5N1 (NIBRG-14) virus. After challenge, the survival wasrecorded for 14 days. (FIG. 5D) The binding of rabbit antiserum fromHA_(mg) with different HAs by ELISA. The rabbit antiserum againstHA_(mg) demonstrated strong binding to HS (CHAS and Vietnam/1194). Inaddition, interactions with H5 (Anhui and ID5/2005) and H1 (NewCaledonia/1999) are also observed.

FIGS. 6A-6C show construction of the H5 HA protein, purification, andgel-filtration chromatography analysis. (FIG. 6A) The DNA encoding theectodomain of HA with cleavage-site alternation was cloned into themammalian expression vector, pTT (Durocher Y, et al. (2002) NucleicAcids Res 30:E9), to allow for efficient secretion of HA proteins fromHEK293 cell cultures. The original protease cleavage site of the HA wasmutated to PQRERG (SEQ ID NO: 2) to avoid the processing of the HA0 intoHA1 and HA2. To stabilize the trimeric conformation of the HA proteins,the “foldon” sequence, which is the bacteriophage trimerizing fragment,was engineered into the plasmid construct, and a His-tag was also addedin the COOH terminus for purification purposes. The expression of HAproteins was carried out by transient transfection with the expressionvector. FIG. 6A depicts SEQ ID NO: 20. (FIG. 6B) The purified HAproteins were analyzed by SDS/PAGE.M indicates marker. Lane 1: HA_(fg),the HA purified from 293E cells; lane 2: HA_(ds), HA_(fg) digested byNA; lane 3: HA_(hm), the HA purified from 293S cells (Reeves P J, et al.(2002) Proc Natl Acad Sci USA 99:13419-13424); lane 4: HA_(mg), HA_(hm)digested by Endo H. (FIG. 6C) The HA-purified proteins were analyzed bygel-filtration chromatography. The eluted peak showed the HA_(fg)trimer>200 kDa (red line), the HA_(ds) trimer>200 kDa (black line), theHA_(hm) trimer>200 kDa (blue line), and the HA_(mg) trimer>180 kDa aftergel filtration (green line). The figure presents superimposed elutionprofiles of HA proteins overlaid with protein marker (dashed line).

FIGS. 7A-7C show mass spectrometry analysis of permethylated N-glycansfrom different HA proteins. MS analysis of permethylated N-glycans fromdifferent HA proteins. (FIG. 7A) The MALDI-MS profile showed that theN-glycans of HA expressed from HEK293E comprise predominantly corefucosylated, biantennary, triantennary, and tetraantennary complex-typeN-glycan structures, as annotated for the major peaks detected andlisted in Table Si. Assignment is based on composition, with only alimited few further verified by MS/MS analysis. The various degree ofsialylation is a principal feature. (FIG. 7B) With NA treatment, all ofthe signals assigned as sialylated N-glycans (e.g., m/z 2605, 3054,3503, 3864, 4226) were no longer detected, concomitant with an increasein signal intensities for the nonsialylated triantennary andtetraantennary structures (m/z 2693, 3142), fully consistent withcomplete removal of the sialic acids. (FIG. 7C) The MALDI-MS profile ofthe N-glycans derived from HA expressed in the GnTI1-deficient HEK293Sstrain showed predominantly a signal corresponding to ManSGlcNAc2 at m/z1579, along with minor peaks of incompletely trimmed high-mannose-typeN-glycans (m/z 1783 to 2396; Hex6HexNAc2-Hex9HexNAc2) in theglycosylation pathway.

FIGS. 8A-8D show MALDI-TOF analysis of HA variants. The molecularweights of (FIG. 8A) HA_(fg) is 75 186.343, (FIG. 8B) HA_(ds) is75290.023, (FIG. 8C) HA_(hm) is 693 14.645 and (FIG. 8D) HA_(mg) is63314.761.

FIG. 9 shows assignments of major molecular ions observed in MALDIspectra of permethylated N-glycans from HA trimers. ND indicates notdetermined.

FIG. 10 shows the ΔΔG of HA glycosylated variants in response to α2,3sialosides 1-15. Values represent ΔΔG, kcal/mol. The entries in theleftmost column were obtained by subtraction of −G values of the latterHA from the former HA (e.g., ΔΔG(HA_(fg)→HA_(ds))=ΔG(HA_(ds))−ΔG(HA_(fg))). ND indicates not determined.

FIG. 11 shows differences in binding free energy changes betweendifferent sialoside ligands in response to HA variants. Values representΔΔG, kcal/mol. The entries in the leftmost column were obtained bysubtraction of ΔG values of the latter HA from the former HA (e.g., ΔΔG(1→2)=ΔG(2)−ΔG(1). ND indicates not determined.

FIG. 12 shows the ability of antisera generated from fully glycosylated,high-mannose and monoglycosylated H5 HA, to inhibit Vietnam 1203 HApseudotyped virus transduction into HEK293 cells. Both high-mannose andmono-glycosylated HA antisera inhibit virus entry, but fullyglycosylated HA antisera does not.

FIGS. 13A-13B show the results of hemagglutination assay with rabbitHA_(fg) and HA_(mg) antisera. FIG. 13A shows results with fullyglycosylated consensus H5 HA antisera in hemagglutination assays towardsH1, H3 and H5. FIG. 13B shows the results with monoglycosylated H5 HAantisera. The monoglycosylated H5 HA antisera not only display goodhemagglutination inhibition activity towards H5, but also towards H1. H3hemagglutination is unaffected by either antisera.

FIG. 14 shows that the protein structures of H5 and H1 are more similarto each other (root mean square deviation (RMSD) of 0.9 Å), than to H3(root mean square deviation (RMSD) of 2.5 Å).

FIGS. 15A-15C show inhibition of NIBRG-121 (Pandemic 2009 A(H1N1)vaccine strain) by antisera generated using mono-glycosylated H1(Brisbane) HA as antigen. FIG. 15A shows inhibition of the ability ofthe NIBRG-121 (Pandemic 2009 A(H1N1) vaccine strain) virus toagglutinate red blood cells. FIG. 15B shows inhibition of the ability ofthe NIBRG-121 (Pandemic 2009 A(H1N1) vaccine strain) virus to infectMDCK cells. FIG. 15C shows protection of BALB/c mice from infection byNIBRG-121 (Pandemic 2009 A(H1N1) vaccine strain) influenza virus. Theantisera used was mice immunized with Brisbane HA proteins (5 μg) andthe virus used for challenge was NIBRG-121 (100×LD₅₀)

FIGS. 16A-16C show inhibition of WSN (H1N1) 1933 by antisera generatedusing mono-glycosylated H1 (Brisbane) HA as antigen. FIG. 16A showsinhibition of the ability of the WSN (H1N1) 1933 virus to agglutinatered blood cells. FIG. 16B shows inhibition of the ability of the WSN(H1N1) 1933 virus to infect MDCK cells. FIG. 16C shows protection ofBALB/c mice from infection by WSN (H1N1) 1933 influenza virus. Theantisera used was mice immunized with Brisbane HA proteins (5 μg) andthe virus used for challenge was WSN (H1N1) 1933 (100×LD₅₀)

FIGS. 17A-17C show inhibition of A/Puerto Rico/8/34 (H1N1): PR8 byantisera generated using mono-glycosylated H1 (Brisbane) HA as antigen.FIG. 17A shows inhibition of the ability of the PR8 virus to agglutinatered blood cells. FIG. 17B shows inhibition of the ability of the PR8virus to infect MDCK cells. FIG. 17C shows protection of BALB/c micefrom infection by PR8 influenza virus. The antisera used was miceimmunized with Brisbane HA proteins (5 μg) and the virus used forchallenge was PR8 (100×LD₅₀)

FIGS. 18A-18B show inhibition of WSN (H1N1) by antisera generated usingmono-glycosylated H1 (Pandemic 2009 A(H1N1) vaccine strain; shown infigure as California/2009) HA as antigen. FIG. 18A shows inhibition ofthe ability of the WSN (H1N1) virus to agglutinate red blood cells. FIG.18B shows inhibition of the ability of the WSN (H1N1) virus to infectMDCK cells.

FIG. 19 shows structural comparison of the glycosylation sites on the H1HA protein.

FIGS. 20A-20B show Dengue type 3 virus envelope glycoprotein E dimers infully glycosylated form (FIG. 20A) and mono-glycosylated form (FIG.20B). Models were created from PDB code 1UZG, with 4 possiblecomplex-type N-glycans with GlyProt server.

FIGS. 21A-21B show human immunodeficiency virus envelope glycoproteingp120 triimers in fully glycosylated form (FIG. 21A) andmono-glycosylated form (FIG. 21B). Models were created from PDB code2BF1, with 13 possible complex-type N-glycans per monomer.

DETAILED DESCRIPTION OF THE INVENTION

In the following detailed description of embodiments of the presentdisclosure, reference is made to the accompanying drawings in which likereferences indicate similar elements, and in which is shown by way ofillustration specific embodiments in which the present disclosure may bepracticed. These embodiments are described in sufficient detail toenable those skilled in the art to practice the present disclosure, andit is to be understood that other embodiments may be utilized and thatlogical, structural, functional, and other changes may be made withoutdeparting from the scope of the present disclosure. The followingdetailed description is, therefore, not to be taken in a limiting sense.

Hemagglutin (HA) of influenza virus is a homotrimeric transmembraneprotein with an ectodomain composed of a globular head and a stemregion. Both regions carry N-linked oligosaccharides, the biosynthesisof which follows the general pathways of N glycosylation. The functionalproperties of HA are affected by glycosylation at specific sites. Thecarbohydrates around the antigenic peptide epi topes interfere with theaccess of antibodies, and this effect may result in antigenic drift ofinfluenza virus. Previous studies on HAs also revealed that the peptidesequences with glycosylation are highly conserved, and the HA receptorbinding specificity was affected by the absence of a complex glycanchain near the receptor binding site. In addition, the proteolyticactivation of HA was also modulated by the glycans near the cleavagesite to influence the infectivity of influenza virus. The extensivevariations in structure and number of glycosylation sites on the headregion have been shown among different subtypes of the influenza Aviruses, whereas the stem oligosaccharides were more conserved andrequired for fusion activity. All these findings have indicated theimportance of HA glycosylation on its activity.

Viral transmission begins with a critical interaction betweenhemagglutinin (HA) glycoprotein, which is on the viral coat ofinfluenza, and sialic acid (SA) containing glycans, which are on thehost cell surface. To elucidate the role of HA glycosylation in thisimportant interaction, various defined HA glycoforms were prepared, andtheir binding affinity and specificity were studied by using a syntheticSA microarray. Truncation of the N-glycan structures on HA increased SAbinding affinities while decreasing specificity toward disparate SAligands. The contribution of each monosaccharide and sulfate groupwithin SA ligand structures to HA binding energy was quantitativelydissected. It was found that the sulfate group adds nearly 100-fold(2.04 kcal/mol) in binding energy to fully glycosylated HA, and so doesthe biantennary glycan to the monoglycosylated HA glycoform. Antibodiesraised against HA protein bearing only a single N-linked GlcNAc at eachglycosylation site showed better binding affinity and neutralizationactivity against influenza subtypes than the fully glycosylated HAselicited. Thus, removal of structurally nonessential glycans on viralsurface glycoproteins is a very effective and general approach forvaccine design against influenza and other human viruses.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994), provides one skilled in the art with a general guide to manyof the terms used in the present application.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

The terms “influenza A subtype” or “influenza A virus subtype” are usedinterchangeably, and refer to influenza A virus variants that arecharacterized by a hemagglutinin (H) viral surface protein, and thus arelabeled by an H number, such as, for example, H1, H3, and H5. Inaddition, the subtypes may be further characterized by a neuraminidase(N) viral surface protein, indicated by an N number, such as, forexample, N1 and N2. As such, a subtype may be referred to by both H andN numbers, such as, for example, H1N1, H5N1, and H5N2. The termsspecifically include all strains (including extinct strains) within eachsubtype, which usually result from mutations and show differentpathogenic profiles. Such strains will also be referred to as various“isolates” of a viral subtype, including all past, present and futureisolates. Accordingly, in this context, the terms “strain” and “isolate”are used interchangeably. Subtypes contain antigens based upon aninfluenza A virus. The antigens may be based upon a hemagglutinin viralsurface protein and can be designated as “HA antigen”. In someinstances, such antigens are based on the protein of a particularsubtype, such as, for example, an H1 subtype and an H5 subtype, whichmay be designated an H1 antigen and an H5 antigen, respectively.

As used in the present disclosure, the term “deglycosylated” or“partially glycosylated” protein denotes a protein that has one or moresugars removed from the glycan structure of a fully glycosylatedinstance of the protein and in which the protein substantially retainsits native conformation/folding. A “deglycosylated” protein includes apartially glycosylated protein in which the deglycosylation processleaves a monoglycosylation, a diglycosylation or a triglycosylation atone or more glycosylation sites present on the glycoprotein.

A “partially glycosylated” protein includes a “deglycosylated” proteinin which one or more sugars are retained at each glycosylation site, andeach partial glycosylation site contains a smaller glycan structure(containing fewer sugar units) as compared to the site in a fullyglycosylated instance of the glycoprotein, and the partiallyglycosylated protein substantially retains its nativeconformation/folding. A “partially glycosylated” protein is generated bypartial deglycosylation of the glycan structure of at least oneglycosylation site of a fully glycosylated instance of the glycoprotein.A “partially glycosylated” protein also is generated by introducingglycosylation at an unglycosylated site of a protein such that the addedglycosylation sequence is smaller than the glycan structure at that sitein a fully glycosylated instance of the glycoprotein. A “partiallyglycosylated” protein also is generated by synthesizing a viralglycoprotein sequence, or fragment thereof, introducing glycosylatedamino acid units (e.g., GlcNAc-Arginine moieties) at glycosylation sitesof the sequence, such that the added glycan structure is smaller thanthe glycan structure at that site in a fully glycosylated instance ofthe glycoprotein.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to refer to single- or double-stranded RNA, DNA, or mixedpolymers. Polynucleotides may include genomic sequences, extra-genomicand plasmid sequences, and smaller engineered gene segments thatexpress, or may be adapted to express polypeptides.

An “isolated nucleic acid” is a nucleic acid that is substantiallyseparated from other genome DNA sequences as well as proteins orcomplexes such as ribosomes and polymerases, which naturally accompany anative sequence. The term embraces a nucleic acid sequence that has beenremoved from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analoguesor analogues biologically synthesized by heterologous systems. Asubstantially pure nucleic acid includes isolated forms of the nucleicacid. Of course, this refers to the nucleic acid as originally isolatedand does not exclude genes or sequences later added to the isolatednucleic acid by the hand of man.

The term “polypeptide” is used in its conventional meaning, i.e., as asequence of amino acids. The polypeptides are not limited to a specificlength of the product. Peptides, oligopeptides, and proteins areincluded within the definition of polypeptide, and such terms may beused interchangeably herein unless specifically indicated otherwise.This term also does not refer to or exclude post-expressionmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring. A polypeptide may be an entire protein, or asubsequence thereof. Particular polypeptides of interest in the contextof this invention are amino acid subsequences comprising CDRs and beingcapable of binding an antigen or HIV-infected cell.

An “isolated polypeptide” is one that has been identified and separatedand/or recovered from a component of its natural environment. Inpreferred embodiments, the isolated polypeptide will be purified (1) togreater than 95% by weight of polypeptide as determined by the Lowrymethod, and most preferably more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS-PAGE under reducing or non-reducing conditions usingCoomassie blue or, preferably, silver stain. Isolated polypeptideincludes the polypeptide in situ within recombinant cells since at leastone component of the polypeptide's natural environment will not bepresent. Ordinarily, however, isolated polypeptide will be prepared byat least one purification step.

A “native sequence” polynucleotide is one that has the same nucleotidesequence as a polynucleotide derived from nature. A “native sequence”polypeptide is one that has the same amino acid sequence as apolypeptide (e.g., antibody) derived from nature (e.g., from anyspecies). Such native sequence polynucleotides and polypeptides can beisolated from nature or can be produced by recombinant or syntheticmeans.

A polynucleotide “variant,” as the term is used herein, is apolynucleotide that typically differs from a polynucleotide specificallydisclosed herein in one or more substitutions, deletions, additionsand/or insertions. Such variants may be naturally occurring or may besynthetically generated, for example, by modifying one or more of thepolynucleotide sequences of the invention and evaluating one or morebiological activities of the encoded polypeptide as described hereinand/or using any of a number of techniques well known in the art.

A polypeptide “variant,” as the term is used herein, is a polypeptidethat typically differs from a polypeptide specifically disclosed hereinin one or more substitutions, deletions, additions and/or insertions.Such variants may be naturally occurring or may be syntheticallygenerated, for example, by modifying one or more of the abovepolypeptide sequences of the invention and evaluating one or morebiological activities of the polypeptide as described herein and/orusing any of a number of techniques well known in the art.

Modifications may be made in the structure of the polynucleotides andpolypeptides of the present invention and still obtain a functionalmolecule that encodes a variant or derivative polypeptide with desirablecharacteristics. When it is desired to alter the amino acid sequence ofa polypeptide to create an equivalent, or even an improved, variant orportion of a polypeptide of the invention, one skilled in the art willtypically change one or more of the codons of the encoding DNA sequence.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of its ability tobind other polypeptides (e.g., antigens) or cells. Since it is thebinding capacity and nature of a protein that defines that protein'sbiological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, its underlying DNAcoding sequence, and nevertheless obtain a protein with like properties.It is thus contemplated that various changes may be made in the peptidesequences of the disclosed compositions, or corresponding DNA sequencesthat encode said peptides without appreciable loss of their biologicalutility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like. Eachamino acid has been assigned a hydropathic index on the basis of itshydrophobicity and charge characteristics (Kyte and Doolittle, 1982).These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5). It is known in the art that certainamino acids may be substituted by other amino acids having a similarhydropathic index or score and still result in a protein with similarbiological activity, i.e. still obtain a biological functionallyequivalent protein. In making such changes, the substitution of aminoacids whose hydropathic indices are within ±2 is preferred, those within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101 states that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with a biological property of the protein. As detailed inU.S. Pat. No. 4,554,101, the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent, and inparticular, an immunologically equivalent protein. In such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Polypeptides may comprise a signal (or leader) sequence at theN-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. A “sequon” is a sequence of threeconsecutive amino acids in a protein that can serve as the attachmentsite to a polysaccharide (sugar) called an N-linked-Glycan. This is apolysaccharide linked to the protein via the nitrogen atom in the sidechain of asparagine (Asn). A sequon is either Asn-X_(aa)-Ser orAsn-X_(aa)-Thr, where X_(aa) is any amino acid except proline. Thus, thepresence of either of these tripeptide sequences in a polypeptidecreates a potential glycosylation site. O-linked glycosylation refers tothe attachment of one of the sugars N-aceylgalactosamine, galactose, orxylose to a hydroxyamino acid, most commonly serine or threonine,although 5-hydroxyproline or 5-hydroxylysine may also be used. While thesequon Asn-X-Ser/Thr is absolutely required for the attachment ofN-linked oligosaccharides to a glycoprotein (Marshall R D, BiochemicalSociety Symposia 40, 17-26 1974), its presence does not always result inglycosylation and some sequons in glycoproteins can remainunglycosylated. (Curling E M, et al., Biochemical Journal 272, 333-3371990)

Glycan microarray is a powerful tool for investigatingcarbohydrate—protein interactions and provides a new platform forinfluenza virus subtyping (Blixt O, et al. (2004) Proc Natl Acad Sci USA101:17033-17038; Chandrasekaran A, et al. (2008) Nat Biotechnol26:107-113; Liang P H, et al. (2007) J Am Chem Soc 129:11177-11184;Stevens J, et al. (2008) J Mol Biol 381:1382-1394). They mimic theglycans on the cell surface to exhibit multivalent interactions withhigh affinity and specificity. Using this technology, characterizationof the receptor specificity of various native and mutant HAs providing anew platform for differentiating influenza virus subtypes was performed.

Although a powerful method, understanding HA-glycan interactions byglycan array analysis has been complicated by two issues: First, HAbinding specificity is affected by the spatial arrangement andcomposition of the arrayed glycans and the binding detection method used(Srinivasan A, et al. (2008) Proc Natl Acad Sci USA 105:2800-2805).Second, the changes in the peptide sequence at or near glycosylationsites may alter HA's 3D structure, and thus receptor-binding specificityand affinity. Indeed, HAs from different H5N1 subtypes have differentglycan-binding patterns (Stevens J, et al. (2008) J Mol Biol381:1382-1394). Mutagenesis of glycosylation sites on H1 and H3 has beenstudied in the whole-viral system (Chandrasekaran A, et al. (2008) NatBiotechnol 26:107-113; Deom C M, et al. (1986) Proc Natl Acad Sci USA83:3771-3775). However, it is not known how changes in glycosylationaffect receptor-binding specificity and affinity, especially with regardto the most pathogenic H5N1 HA. To address these issues, a method ofquantitative glycan microarray analysis was developed to surmount thelimitations of traditional HA binding experiments.

Previous studies have used HA from insect cell expression(Chandrasekaran A, et al. (2008) Nat Biotechnol 26:107-113). However,glycosylation in insect cells differs from mammalian cells, with amarked difference being that complex type N-glycans terminating ingalactose and sialic acid are not produced in insect cells.

HA Glycosylated Variants Expressed from Human Cells

To address how changes in glycosylation affect receptor-bindingspecificity and affinity in human cells, a glycan microarray comprisingextensive structural analogs of the HA-binding ligand, and severaldefined glycoforms of HA were prepared by using the influenza H5N1 HAconsensus sequence (Chen M W, et al. (2008) Proc Natl Acad Sci USA105:13538-13543) for quantitative binding analysis.

The codons of CHAS were optimized for expression by using human codons.As shown in Table 1, the original viral protease cleavage sitePQRERRRKKRG (SEQ ID NO: 1) was mutated to PQRERG (SEQ ID NO: 2) in orderto prevent proteins from the enzymatic cleavage to form HA1 and HA2. Thetransmembrane region (residues: 533-555) was replaced with theadditional residues

(SEQ ID NO: 3) LVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG HHHHHHat the C terminus of the HA construct, where the thrombin cleavage siteis in italics, the bacteriophage T4 fibritin foldon trimerizationsequence is underlined, and the His-tag is in bold (Stevens J. et al.(2006) Science 312:404-410).

TABLE 1 Consensus H5 hemagglutinin sequenceAmino acid sequence of consensus H5 HA showing in bold: signalsequence; and underlined: trimerization sequence and His-tag.MEKIVLLFAIVSLVKSDQICI GSHANNSTEQVDTIMEKNVTVTHAQDILE  50KTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKAN 100PANDLCYPGDFNDYEELEHLLSRINHFEKIQIIPKSSWSSHEAGSGVSSA 150CPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDA 200AEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILK 250PNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGA 300INSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERGLFGAIAGF 350IEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKM 400NTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLD 450FHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDY 500PQYSEEARLKREEISGVDIRSLVPRGSPGSGYIPEAPRDGQAYVRKDGEW 550 VLLSTFLGHHHHHH(SEQ ID NO: 4) Nucleotide sequence of consensus H5 HA    1ATGGAGAAGA TCGTGGTGCT GTTCGCCATC GTGAGCCTGG TGAAGAGCGA   51CCAGATCTGC ATCGGATCCC ACGCCAACAA CAGCACCGAG CAGGTGGACA  101CCATCATGGA GAAGAACGTG ACCGTGACCC ACGCCCAGGA CATCCTGGAG  151AAGACCCACA ACGGCAAGCT GTGCGACCTG GACGGCGTGA AGCCTCTGAT  201CCTGAGAGAC TGCAGCGTGG CCGGCTGGCT GCTGGGCAAC CCTATGTGCG  251ACGAGTTCAT CAACGTGCCT GAGTGGAGCT ACATCGTGGA GAAGGCCAAC  301CCTGCCAACG ACCTGTGCTA CCCTGGCGAC TTCAACGACT ACGAGGAGCT  351GAAGCACCTG CTGAGCAGAA TCAACCACTT CGAGAAGATC CAGATCATCC  401CTAAGAGCAG CTGGAGCAGC CACGAGGCCA GCAGCGGCGT GAGCAGCGCC  451TGCCCTTACC AGGGCAAGAG CAGCTTCTTC AGAAACGTGG TGTGGCTGAT  501GAAGAAGAAC AGCACCTACC CTACCATCAA GAGAAGCTAC AACAACACCA  351ACCAGGAGGA CCTGCTGGTG CTGTGGGGCA TCCACCACCC TAACGACGCC  601GCCGAGCAGA CCAAGCTGTA CCAGAACCCT ACCACCTACA TCAGCGTGGG  651CACCAGCACC CTGAACCAGA GACTGGTGCC TAAGATCGGC ACCAGAAGCA  701AGGTGAACGG CCAGAGCGGC AGAATGGAGT TCTTCTGGAC CATCCTGAAG  751CCTAACGACG CCATCAACTT CGAGAGCAAC GGCAACTTCA TCGCCCCTGA  801GTACGCCTAC AAGATCGTGA AGAAGGGCGA CAGCACCATC ATGAAGAGCG  851AGCTGGAGTA CGGCAACTGC AACACCAAGT GCCAGACCCC TATGGGCGCC  901ATCAACAGCA GCATGCCTTT CCACAACATC CACCCTCTGA CCATCGGCGA  951GTGCCCTAAG TACGTGAAGA GCAACAGACT GGTGCTGGCC ACCGGCCTGA 1001GAAACAGCCC TCAGAGAGAG AGAGGCCTGT TCGGCGCCAT CGCCGGCTTC 1051ATCGAGGGCG GCTGGCAGGG CATGGTGGAC GGCTGGTACG GCTACCACCA 1101CAGCAACGAG CAGGGCAGCG GCTACGCCGC CGACAAGGAG AGCACCCAGA 1151AGGCCATCGA CGGCGTGACC AACAAGGTGA ACAGCATCAT CGACAAGATG 1201AACACCCAGT TCGAGGCCGT GGGCAGAGAG TTCAACAACC TGGAGAGAAG 1251AATCGAGAAC CTGAACAAGA AGATGGAGGA CGGCTTCCTG GACGTGTGGA 1301CCTACAACGC CGAGCTGCTG GTGCTGATGG AGAACGAGAG AACCCTGGAC 1351TTCCACGACA GCAACGTGAA GAACCTGTAC GACAAGGTGA GACTGCAGCT 1401GAGAGACAAC GCCAAGGAGC TGGGCAACGG CTGCTTCGAG TTCTACCACA 1451AGTGCGACAA CGAGTGCATG GAGAGCGTGA GAAACGGCAC CTACGACTAC 1501CCTCAGTACA GCGAGGAGGC CAGACTGAAG AGAGAGGAGA TCAGCGGCGT 1551GGATATCAGA TCTCTGGTGC CAAGAGGATC TCCAGGATCT GGATACATCC 1601CAGAGGCTCC AAGAGATGGA CAAGCTTACG TGAGAAAGGA CGGAGAGTGG 1651 GTGCTGCTGT CTACTTTCCT GGGACACCAC CACCACCACC ACTAA (SEQ ID NO: 5)

The H5 consensus sequence was used to generate HA glycosylated variantsexpressed from HEK293 human cells. To generate high-mannose-typeglycosylation (HA_(hm)), HEK293S cells, which are deficient inN-acetylglucosaminyltransferase I (GnTI⁻), were used. In order tofurther address the effect of HA glycosylation on receptor bindingaffinity and specificity, the sugars on HA were systematically removedfrom the native complex type N-glycans (FIG. 1A). Sialic acid residueswere removed from HA_(fg) by neuraminidase (NA) treatment to producedesialylated HA (HA_(dS)). Endoglycosidase H (Endo H) was used totruncate all of the glycan structures down to a single GlcNAc residue toproduce monoglycosylated HA (HA_(mg)). Thus, a total of four glycoformsof HA were generated: HAfg: fully glycosylated HA from human HEK293Ecells; HA_(dS) desialylated HA from neuraminidase (NA) treatment ofHA_(fg); HA_(hm): high-mannose type HA from human N-acetylglucosaminyltransferase I deficient (GnTI-) HEK293S cells, and HA_(mg): HA withsingle N-acetyl glucosamine residue at its glycosylation sites fromEndoglucosidase H (Endo H) treatment of HA_(hm) (FIG. 1A and FIG. 6 ).The glycan structures are verified by mass spectral analysis (FIGS. 7,8, 9 ). Circular dichroism of the variants confirmed that theirsecondary structures are similar (FIG. 1C). The sole effect fromN-glycans on HA of different glycoforms were then studied, assuming theprotein 3D structures of these samples are similar and not causing biasin the analysis (FIG. 1B). It is noted that an attempt to expressfunctional HA in Escherichia coli failed because of the lack ofglycosylation.

Moreover, mass spectrometry analyses confirmed that (a) HA_(fg) containspredominantly the complex type N-glycans (FIG. 7A); (b) the sialic acidshave been removed from the complex type N-glycans on HA_(ds) (FIG. 7B);(c) HA_(hm) contains predominantly the high mannose type N-glycans (FIG.7C); and (d) HA_(mg) showed only an N-acetylglucosamine (GlcNAc) on HA(FIG. 1 and FIG. 8 ).

Glycan Microarray Profiling of HA Glycosylated Variants

Glycan microarray profiling of HA glycosylated variants HA_(fg),HA_(ds), HA_(hm), and HA_(mg) were examined by using traditionalsandwich method. The synthetic sialic acid glycan array consisted of 17of the α2,3 (glycans 1-17) and 7 of the α2,6 (glycans 21-27) sialosidesdesigned to explore the glycan specificity of influenza viruses (seeFIG. 4 ). The synthetic sialosides with a five-carbon linker terminatedwith amine were prepared and covalently attached onto NHS-coated glassslides by forming an amide bond under aqueous conditions at roomtemperature. The printing procedure was based on the standard microarrayrobotic printing technology, as reported previously (Blixt O, et al.(2004) Proc Natl Acad Sci USA 101:17033-17038; Wang C C, et al. (2008)Proc Natl Acad Sci USA 105:11661-11666). HA variants were applied to thesialic acid slides and then hybridized with primary antibody, followedby detection with a secondary antibody conjugated to Cy3. This analysisindicated that the H5N1 HA consensus sequence specifically binds to α2,3sialosides but not α2,6 sialosides (FIG. 2A), in accordance withprevious studies (Chandrasekaran A, et al. (2008) Nat Biotechnol26:107-113; Stevens J, et al. (2008) J Mol Biol 381:1382-1394).Surprisingly, the binding strength with α2,3 sialosides grewsuccessively stronger from HA_(fg), HA_(ds), and HA_(hm), to HA_(mg)(FIG. 2A) by qualitative binding via relative fluorescence intensity.

HAs were contacted with a synthetic glycan array containing 17 α2-3(glycan 1-17) and 7 α2-6 (glycan 21-27) sialosides designed forinfluenza virus (FIG. 4 ), and then the HA proteins were hybridized withunlabeled primary followed by detection by Cy3 tagged secondaryantibody. The analysis indicated that influenza HSNI hemagglutinin ofconsensus sequence can specifically bind to α2-3 sialosides but not α2-6sialosides (FIG. 2A). Surprisingly, the binding with α2-3 sialosides wassuccessively stronger from HA_(fg), HA_(ds), HA_(hm), to HA_(mg) (FIG.2A) in the intensity comparison of glycan array profiling.

Quantitative Glycan Microarray

Glycan array profiling has been limited to the qualitative natures inbinding events investigation because it only provides the relativefluorescence intensity, and users cannot differentiate the bindingaffinity to receptors from the separate experiments. In order toprecisely determine the binding events, this microarray platform wasextended to determine the dissociation constants of HA-glycaninteractions quantitatively.

A quantitative array was designed to determine surface dissociationconstants (Liang P H, at el. (2007) J Am Chem Soc 129:11177-11184). Toavoid any skewing by antibody layering, HA was directly labeled with thefluorescent dye Cy3 (Srinivasan A, et al. (2008) Proc Natl Acad Sci USA105:2800-2805). Direct binding assays were performed by serial dilutionof Cy3-labeled HAs to establish the relative binding intensities. Thedissociation constants on the surface were determined by plotting the HAconcentrations against fluorescence intensity for each of the 24sialosides printed on the glass slide. The dissociation constantK_(D,surf) values were calculated based on the Langmuir isotherms (seeFIG. 2B). The monovalent HA-sialoside binding is weak, exhibitingdissociation constants in the millimolar range (K_(D)=2.5×10⁻³ M)(Sauter N K, et al. (1989) Biochemistry 28:8388-8396); however, HA isinvolved in multivalent interactions with sialosides on the host cellsurface, which can be seen in the quantitative array profiling (Table2). The K_(D,surf) values decreased globally and substantially as thelength of N-glycans on HAs decreased (FIG. 2B).

All HA glycoforms showed strong binding to receptor glycans with asulfate group at the 6 position of the third GlcNAc residue from thenonreducing end (glycans 4 and 7). This sulfate group is important forbinding to H5 HA (Chandrasekaran A, et al. (2008) Nat Biotechnol26:107-113; Stevens J, et al. (2008) J Mol Biol 381:1382-1394). Inaddition, it was observed that glycan 4 is the best ligand for HA_(fg),whereas glycans 13-15 are better ligands than glycan 6 for HA_(mg),indicating a possible multivalent interaction within the ligand-bindingsite, or the exposure of more receptor-binding domains to biggerbiantennary sialosides (glycans 13 and 14). Interestingly, HA bindingsubstantially increases as its N-glycan structures become less complex(FIG. 2B). However, although the K_(D,surf) values for HA_(mg) showstronger and similar binding to a few SA glycans, the other HA variantsexhibit weaker and more specific binding to glycan ligands (FIGS. 2B and10 ). Thus, binding specificity and binding affinity may have an inverserelationship that is modulated by glycan structure. This modulation mayhave important biological significance, in that the carbohydrates on HAcan tune its recognition of glycan receptors on the lung epithelialcells.

Binding Energy Contribution from Receptor Sialosides

Kinetic parameters can be applied to thermodynamic parameters toillustrate the interaction events in molecular details. The dissociationconstant (K_(D,surf)) of HA-glycan interactions can be used to calculatethe Gibbs free energy change of binding (ΔG_(multi)). Values forΔG_(multi) represent a quantitative measurement of stabilizing energyfrom HA-glycan interactions. A successive decrease in ΔG_(multi)correlated with the systematic decrease in complexity/truncation of theN-glycan structures on HA (Table 2).

TABLE 2 Dissociation constants (K_(D, surf)) and free energy changes(ΔG) of HA glycosylated variants when binding to α2,3 sialosides 1-15K_(D, surf), μM ± SD ANOVA ΔG, kcal/mol ± SD Sialosides HA_(fg) HA_(fg)HA_(fg) HA_(fg) P* HA_(fg) HA_(fg) HA_(fg) HA_(fg) 1 6.99 ± 0.41 2.86 ±0.93 2.09 ± 0.59 0.27 ± 0.16 <0.0001  −7.03 ± 0.03 −7.58 ± 0.19 −7.76 ±0.17 −8.80 ± 0.15 2 3.72 ± 1.01 2.47 ± 0.21 1.75 ± 0.32 0.20 ± 0.070.0002 −7.41 ± 0.16 −7.66 ± 0.06 −7.86 ± 0.11 −9.03 ± 0.07 3 4.55 ± 1.852.34 ± 0.27 0.92 ± 0.16 0.26 ± 0.06 0.0002 −7.31 ± 0.25 −7.68 ± 0.07−8.24 ± 0.10 −8.90 ± 0.01 4 0.27 ± 0.01 0.27 ± 0.05 0.33 ± 0.09 0.13 ±0.06 0.0048 −8.96 ± 0.03 −8.95 ± 0.10 −8.84 ± 0.16 −9.45 ± 0.27 5 ND5.20 ± 1.01 9.40 ± 3.20 0.54 ± 0.15 ND ND −7.21 ± 0.11 −6.88 ± 0.21−8.49 ± 0.13 6 20.03 ± 4.24  9.22 ± 2.05 2.71 ± 0.53 0.80 ± 0.05<0.0001  −6.41 ± 0.13 −6.87 ± 0.13 −7.65 ± 0.06 −8.32 ± 0.05 7 0.57 ±0.10 0.77 ± 0.08 0.61 ± 0.02 0.32 ± 0.10 0.0010 −8.46 ± 0.06 −8.36 ±0.04 −8.47 ± 0.02 −8.78 ± 0.14 8 2.49 ± 0.58 2.48 ± 0.41 1.69 ± 0.530.36 ± 0.13 0.0008 −7.65 ± 0.14 −7.65 ± 0.10 −7.89 ± 0.21 −8.82 ± 0.30 9ND 15.34 ± 5.06  4.40 ± 0.56 0.86 ± 0.34 ND ND −6.58 ± 0.20 −7.31 ± 0.08−8.18 ± 0.16 10 7.64 ± 2.3  3.61 ± 0.61 1.22 ± 0.52 0.29 ± 0.14 0.0003−6.99 ± 0.18 −7.43 ± 0.10 −8.09 ± 0.24 −8.77 ± 0.03 11 6.02 ± 1.04 2.32± 0.14 1.11 ± 0.51 0.33 ± 0.08 <0.0001  −7.12 ± 0.10 −7.68 ± 0.04 −8.15± 0.25 −8.91 ± 0.18 12 40.23 ± 9.77  ND 2.45 ± 0.52 1.41 ± 0.92 ND −6.00± 0.15 ND −7.66 ± 0.12 −7.85 ± 0.25 13 3.38 ± 1.06 1.37 ± 0.30 0.31 ±0.06 0.07 ± 0.01 0.0008 −7.47 ± 0.19 −8.05 ± 0.13 −8.88 ± 0.13 −9.77 ±0.09 14 2.72 ± 0.41 0.97 ± 0.41 0.42 ± 0.03 0.09 ± 0.01 <0.0001  −7.59 ±0.09 −8.27 ± 0.28 −8.69 ± 0.04 −9.60 ± 0.01 15 2.37 ± 0.19 1.32 ± 0.160.89 ± 0.35 0.09 ± 0.01 0.0002 −7.67 ± 0.05 −8.02 ± 0.07 −8.29 ± 0.27−9.62 ± 0.08

Table 2 shows thermodynamic parameters of HA with differentglycosylations in response to α2,3 sialosides 1-15. Free energy changes(ΔG) and K_(D,surf) of HA-glycan interactions are shown in response toα2,3 sialosides 1-15. ΔG values can be derived from K_(D,surf) values byusing the equation ΔG_(multi)=−RT ln(K_(D,surf) ⁻¹). The values of ΔGwere calculated according to K_(D,surf) values to obtain free energychanges in HA-glycan binding. ΔG(HA_(fg)) of glycans 5 and 9 is notdetermined. ND indicates not determined. (*From the set of 15 identifiedHA-binding sialosides, statistically significant differences ofK_(D,surf) values among four HA glycoforms are shown by using a one-wayANOVA (P<0.05 is considered significant)).

The differences in free energy change (ΔΔG) between HA variants arecaused by unique glycan structures (FIG. 10 ), and the largestdifference is between HA_(fg) and HA_(mg) (ΔΔG HA_(fg)→HA_(mg); see FIG.10 ), which is consistent with the largest difference in binding energyresulting from trimming off most of the N-glycan down to a singleGlcNAc. It is noted that values of ΔΔG are similar except for glycans 4and 7 (FIG. 10 ), indicating that glycans on HA do not significantlyaffect the binding affinity with sulfated α2,3 trisaccharide(Chandrasekaran A, et al. (2008) Nat Biotechnol 26:107-113).

HA-Receptor Binding

The molecular details of the HA-receptor binding (i.e., the contributionfrom each structural component comprising a glycan receptor) can beaddressed by comparing the differences in free energy change (ΔΔGvalues) between different receptor sialosides (FIGS. 4 and 11 ).Dissecting the energy contribution of the receptor sialosidesresponsible for HA binding reveal key points of specificity that areused to design new HA inhibitors. Sialosides α2,3 linked to galactoseresidues with β1,4 (Galβ1-4) linkages possess better binding affinitythan those with Galβ1-3 linkages (Stevens J, et al. (2008) J Mol Biol381:1382-1394). This is reflected in the comparison of theNeu5Ac-α2,3-galactose (Neu5Acα2,3Gal) disaccharide backbone (FIG. 4A,glycan 1, red box highlight), where trisaccharides 3 and 6 only differin the linkage between Gal and GlcNAc. Here, the ΔΔG(1→3) for all HAvariants is negative (stabilizing HA-receptor interaction), whereas theΔΔG(1→6) for all HA variants is positive (destabilizing HA-receptorinteraction; FIGS. 4A and 11 ). This observation indicates thatNeu5Ac-α2,3Galβ1-4Glc/GlcNAc is the core glycan component interactingwith the HA-binding pocket. Moreover, the value of ΔΔG(1→9) for all HAvariants is positive, indicating a negative perturbation caused by theβ6-linked mannose at the third position (FIGS. 4A and 11 ). Thus,binding energy is affected by inner sugar residues and their linkagepatterns to the distal Neu5Ac-α2,3Gal disaccharide ligand (FIG. 4A).This analysis shows that a GlcNAc residue at the third position isfavored for all HA variants. However, in comparing ΔΔG values forglycans 13 and 14 (FIGS. 4E and 11 ) to glycan 6, multivalentinteractions in the binding site with the biantennary sialoside areapparent, and for HA_(mg), this intramolecular avidity is moresignificant for driving binding than the structural effect exerted bythe third sugar.

Next, receptor glycans 10, 11, 12, and 15 were compared. These have thesame basic core structure (glycan 8 trisaccharide) but differ byelongation (glycans 11 and 12) or addition of an α2,6 sialic acid at thethird position (glycan 15; FIG. 4B). It is interesting that thesialoside with the branched α2,6 sialic acid greatly increased HAavidity, whereas the longer α2,3 sialoside extending from glycan 8resulted in a weaker binding by HAs (ΔΔG (8→15)>ΔΔG (8→11)˜ΔΔG(8→10)>ΔΔG (8→12); FIGS. 4B and 11 ).

Glycans 3-5 and 6-7 share the same trisaccharide backbone but differ bythe addition of a sulfate group (glycan 4) or fucose residue (glycan 5)on the third GlcNAc from the nonreducing end. The sulfate group canstabilize the HA-receptor glycan interaction up to 2.044 kcal/mol (ΔΔG(6→7)), the largest energy gap between two receptor sialosides. Amongall of the HA variants, the fully glycosylated variant showed the mostsignificant differences in free energy changes, with values of ΔΔG(3→4)HA_(fg) (−1.653 kcal/mol) and ΔΔG(6→7) HA_(fg) (−2.044 kcal/mol), andthe size of the free energy gain lessened as the glycan structure becamemore simplified; i.e., HA_(fg)>HA_(ds)>HA_(hm)>HA_(mg). Thus, sulfatedglycans dramatically enhance HA binding, and fully glycosylated HAmaximizes this effect (FIGS. 4C and D), which is important for H5N1pathogenesis. On the other hand, the fucosylated receptor analogsgreatly destabilize HA binding, with all glycosylated HA variantsshowing a positive ΔΔG(3→5) (FIG. 4C). These large differences inΔΔG(3→4) and ΔΔG(3→5) are likely caused by an important bindinginteraction in the receptor-binding pocket, which the sulfate groupmaximizes and the fucose sterically blocks. The weak binding of HA_(fg)is unlikely due to the competition of its sialylglycans, because removalof sialic acid has a small effect on binding, and HA_(fg) still exhibitsa strong affinity for certain specific sialylglycans.

Vaccine Design Using Monoglycosylated HA

The monoglycosylated hemagglutinin HA_(mg) shows a similar secondarystructure and better binding affinity to host receptors as compared toits fully glycosylated counterpart. Recent studies also indicated that asingle GlcNAc residue to Asn is the minimum component of the N-glycanrequired for glycoprotein folding and stabilization (Hanson S R, et al.(2009) Proc Natl Acad Sci USA 106:3131-3136). Because proteins aresuperior immunogens to glycans, the monoglycosylated HA was tested as aprotein vaccine against influenza viruses. Antisera from HA_(fg) andHA_(mg) immunizations were compared with regard to their ability to bindnative HAs and to neutralize H5 viruses (FIG. 5 ). Indeed, in contrastto HA_(fg), the antiserum from HA_(mg) showed stronger neutralization ofthe virus. The HA_(mg) antiserum also binds to H1 (New Caledonia/1999)in addition to the H5 subtypes Vietnam/1194, H5 (Anhui), and H5(ID5/2005) (FIG. 5D). Notably, the HA_(mg) vaccine was much moreprotective than the HA_(fg) vaccine in a challenge study (FIG. 5C).

The amino acid sequences of H1, H3, and H5 isolated from humans werecompared I FIG. 3 . When comparing H1 vs. H3 and H3 vs. H5, differencesin the overall amino acid sequences as well as those nearN-glycosylation sites are observed. H1 and H5 show higher overall aminoacid sequence similarities, and the sequences near N-glycosylation sitesare more conserved. Seasonal (A/Brisbane/59/2007) and Pandemic(A/California/07/2009) H1 strains show about 79% sequence identity. Theoverall sequence identity was about 63% between H1 and H5, and about 40%between both H3 and H5, and H1 and H3. In addition, the N-glycosylationsites (shown within red boxes in FIG. 3 ) and the underlying peptidesequences are more conserved between H1 and H5 than between H1/H3 andH5.

The present invention shows that the systematic simplification ofN-glycans on HA results in a successive increase in binding to α2,3sialosides but not to α2,6 sialosides. The inventors, for the firsttime, show the effect of HA's outer and inner glycans on receptorbinding and to quantitatively dissect the binding affinity and energeticcontributions of HA-receptor interactions.

HA glycosylation affects the function of influenza HA (Wagner R, et al.(2002) J Gen Virol 83:601-609). Interestingly, as the level ofglycosylation on influenza H3N2 has increased since 1968, the morbidity,mortality, and viral lung titers have decreased (Vigerust D J, et al.(2007) J Virol 81:8593-8600).

Without being bound by theory, the finding that HA with a single GlcNAcattached to the glycosylation sites shows relaxed specificity butenhanced affinity to α2,3 sialosides suggests that the N-glycans on HAmay cause steric hindrance near the HA-receptor binding domain. The highspecificity for receptor sialosides may prevent the virus from bindingto some other specific glycans on the human lung epithelial cellsurface. On the other hand, HA with truncated glycans can recognize α2,3receptor sialosides with higher binding affinity and less specificity,suggesting that reducing the length of glycans on HA may increase therisk of avian flu infection. It is, however, unclear how the changes ofHA-receptor interaction via glycosylation affect the infectivity of thevirus and the NA activity in the viral life cycle.

HA with a single GlcNAc is a promising candidate for influenza vaccinebecause such a construct retains the intact structure of HA and can beeasily prepared (e.g., via yeast). It also can expose conserved epitopeshidden by large glycans to elicit an immune response that recognizes HAvariants in higher titer. This strategy opens a new direction forvaccine design and, together with other different vaccine strategies(Hoffmann E, et al. (2005) Proc Natl Acad Sci USA 102:12915-12920;Huleatt J W, et al. (2008) Vaccine 26:201-214; Scanlan C N, et al.(2007) J Mol Biol 372:16-22; Yang Z Y, et al. (2007) Science317:825-828) and recent discoveries of HA-neutralizing antibodies(Ekiert D C, et al. (2009) Science 324:246-251; Kashyap A K, et al.(2008) Proc Natl Acad Sci USA 105:5986-5991; Scheid J F, et al. (2009)Nature 458:636-640; Stevens J, et al. (2006) Science 312:404-410; Sui JH, et al. (2009) Nat Struct Mol Biol 16:265-273), should facilitate thedevelopment of vaccines against viruses such as influenza, hepatitis Cvirus, and HIV.

Therefore, whether HA with a single GlcNAc can be a promising candidatefor influenza vaccine was tested. For the benefits of its strong bindingwith 0.2-3 sialosides, HA with a single GlcNAc can elicit immuneresponse that recognize the region close to RBD with higher titers,indicating that the addition of oligosaccharides can be an effectivemeans of immune evasion via the modification or masking of antigenicepitopes on the virus. Therefore, the strategy of removal of mostglycans, hut with at least a single GluNAc retaining opens a newdirection for future vaccine design, and this concept provides insightinto other anti-virus vaccine design such as HCV, HBV, and HIV. Otheriterations have two, three, or more glycans of the original glycan chainremaining.

Partially Glycosylated Cell-Surface Glycoproteins as Vaccines

The cell-surface glycoproteins of viruses are good targets for vaccinedevelopment. However, such surface proteins are often highlyglycosylated by the host to protect the virus from the host's immunesystem. In addition, the viral protein sequences around theglycosylation sites are often highly conserved and thus are good antigenfor the vaccine design, however, these highly conserved regions are notreadily available to the host's immune system at least in part due tothe amount of glycosylation covering or blocking those regions. Forexample, one reason for the limited success in the preparation ofvaccines against intact HIV is because the viral surface gp120 is highlyglycosylated.

The new vaccine is more immunogenic and the antibody induced is expectedto have better neutralization activity against the intact glycoprotein,which is made by the virus and the host. The antibody is able to attackboth the less or non-glycosylated region(s) which is more likely tomutate and the glycosylated region which is highly conserved, lesslikely to mutate and/or sensitive to mutation. An antibody generatedthusly will strongly interact with the protein part of the target assuch antibody has higher affinity for protein than carbohydrate and thusthermodynamically it will push the glycan chain away to bind the highlyconserved regions around the glycosylation sites.

In the O- and N-linked glycoproteins, the first sugar(N-acetylglucosamine for N-glycoprotein and N-acetylglucosamine orN-acetylgalactosamine for O-glycoproteins) is essential to preserve thetertiary structure of the glycoprotein while the rest of the sugars arenot important. Treatment of N-glycoproteins with the endoglycosidase(endoH) will remove the sugar chain and keep the N-acetyl glucosamineattached to the protein. Mannosidases may also be used to cleaveN-glycoproteins to di- or triglycans, which are expressly contemplatedherein as possible vaccines due to the ability of the immune system toaccess the conserved glycosylation sites on the proteins even with di-,tri-, and larger deglycosylated proteins. Other glycosidases are alsoavailable to remove the sugar chain from O-glycoproteins and keep thefirst sugar attached to the protein.

When a fully glycosylated hemagglutinin (HA) from bird flu (H5)expressed in human cells is treated with endoH to reduce glycosylationto a monoglycosylated state and used in the immunization of rabbit, theantiserum generated has a higher titer than the antiserum generated fromthe fully glycosylated hemagglutinin. (FIGS. 13A-13B). It is also ableto neutralize the hemagglutinin from other bird flu strains and thehemagglutinin H1 from human flu while the antiserum from the fullyglycosylated HA cannot neutralize H1 and is more specific for the birdflu strain.

The similarity of glycosylation pattern and protein structure between H1and H5 provides a possible reason why the mono-glycosylated H5 antiserumcross-reacts with H1. The data was obtained using rabbit antisera. (FIG.13 ). As shown in FIGS. 12-14 , the hemagglutinin of H3 does not havethe same degree of homology as H5 HA does with H1 HA. Thus, antiserumgenerated from H1 and H5 do not neutralize H3. However, because H3shares other homology in the conserved regions with H1 and H5, antiserumgenerated from deglycosylated hemagglutinin H3 neutralizes hemagglutininH1 and hemagglutinin H5 in addition to hemagglutinin H3.

To prepare monoglycosylated hemagglutinin, it is not necessary to makethe glycoprotein from human cell culture as the glycoprotein with thefirst three sugars (Mannose-N-acetylglucosamine-N-acetylglucosamine) oronly the monosaccharide (N-acetylglucosamine) attached to the protein(i.e., N-acetylglucosamine-protein) is highly conserved in eukaryotes.Thus, one can make the glycoprotein in yeast, baculovirus, or othereukaryotic hosts and treat the glycoprotein mixture with the appropriateglycosidase, such as endoH or mannosidase for N-linked glycoproteins, toprepare the homogeneous monoglycosylated protein for use as vaccine.

Without being bound by theory, it is postulated that N-linked glycan ismuch longer than the O-linked glycan and it is the N-linked glycan thatneeds to be trimmed to remove the rest of sugar chains. Therefore, theO-linked glycan will not cause a problem even if it is intact.

A native glycoprotein that is exposed to the immune system has anN-linked branched glycoprotein. Because of the glycosylation, the highlyconserved region is inaccessible to the immune system. Thus, the immunesystem can only target highly variable regions, thereby reducing thesubject susceptible to multiple viral infections as the variable regionsmutate. If the immune system is able to access the highly conservedregions, then antibodies directed to these sequences which do not varyover time provide a route towards inoculation against viruses thateither have variable regions that mutate and thereby render existingantibodies against the glycoproteins ineffective or are so thicklyglycosylated as to be substantially inaccessible to the immune system.

Therefore, to make the protein accessible to the immune system via avaccine, the glycosylation is removed, thereby exposing the native viralprotein to the immune system. Importantly, complete removal of thesugars from the protein has been shown to cause the protein to denature;in many viral glycoproteins, glycosylation is a key component totertiary structure of the glycoprotein.

The sugars are removed by exposing the isolated native glycosylatedproteins to a N-glycosidase, for example endoH or mannosidase, whichwill cleave all but the first one, two, or three sugars from theglycoprotein, without causing the protein to lose its tertiarystructure. The deglycosylated proteins are then formulated with asuitable pharmaceutical carrier as a vaccine and administered to asubject. Because the highly conserved glycosylation regions are nowdeglycosylated and thereby exposed to the immune system, antibodies aregenerated against the highly conserved regions.

When the immunized subjects are infected with the virus and the viralglycoproteins are exposed to the immune system, the antibodies that aredirected to the highly conserved regions of the protein are present inthe subject's system. Thus, mutation in variable regions becomesirrelevant because there are still antibodies directed to thenon-mutating conserved regions of the glycoprotein.

Moreover, glycosylation does not hinder binding of the antibodies to thehighly conserved regions because the antibodies are thermodynamicallyinclined to bind the protein and “push” the sugars out of the way forbinding of the antibodies to the highly conserved regions. Importantly,in viral proteins such as gp120 of HIV, this strategy provides a methodand composition for inoculation where the immune system would otherwisenot produce an antibody titer large enough to effectively fight theinfection.

According to implementations embodying these principles, a vaccinecomprising at least one deglycosylated hemagglutinin and apharmaceutically acceptable carrier is contemplated. The vaccine can bemade using any system that expresses glycosylated proteins, such asyeast and baculovirus. Once the proteins are made, they are isolatedusing a suitable method, such as gel electrophoresis, chromatography, orother methods capable of isolating proteins.

The pattern of glycosylation at the glycosylation site is conserved innearly all eukaryotes (GlcNAc-GlcNAc-Man). Thus, it doesn't matter whatthe downstream glycosylation pattern is, provided that the first 1-3(potentially more depending on the organism being inoculated and theorganism producing the protein) sugars remain. Thus, for a humanvaccine, yeast may be used to produce the protein used in a humanvaccination, because once all but the first one to three sugars arecleaved, the pattern is identical to the first one to three sugars inthe human version of the glycoprotein. Therefore, the present disclosureprovides a unique platform for high throughput, high output productionof vaccines against virus such as influenza, HIV, and flavivirus.

To generate the vaccines, the glycosylated proteins are isolated andthen (partially) deglycosylated using a glycosidase, or another enzymeor method that selectively digests the carbohydrates forming theglycosylation. However, whatever method is used to cleave the sugarchains it must not affect the tertiary structure of the underlyingprotein.

The partially glycosylated glycoproteins, or fragments thereof, also canbe prepared synthetically. There are two strategies for the synthesis ofglycopeptides. (i) Stepwise method: glycosylamino acids are used as abuilding block for solid-phase synthesis. The advantage of this approachis “wide use” for the preparation of various glycopeptides. Thisapproach allows the preparation of glycopeptides having someoligosaccharide moieties. (ii) Convergent method: an oligosaccharidemoiety and a peptide moiety are prepared separately, then coupled witheach other. Commonly, this approach is used for the preparation ofN-glycopeptides. This approach requires a special orthogonal side-chainprotecting group for the “glycosylation point” in the peptide moiety.

In one embodiment, N-acetylglucosamine (GlcNAc) attached to theasparagine residue of the peptide can be synthesized using a thioestermethod to build the polypeptide segment (Merrifield R B. J. Am. Chem.Soc. 85:2149 (1983)) and a dimethylphosphinothioic mixed anhyride(Mpt-MA) method for the incorporation of the glycopeptide moiety (Guo, ZW, et al. (1997) Angew. Chem. Int. Ed. Engl. 36, 1464-1466).

Cross-Reactive Influenza Vaccines Generated from Mono-Glycosylated HAProteins

(A) Vaccination with Seasonal H1 (Brisbane) Mono-Glycosylated HAProtein.

A hemagglutination inhibition assay was used to detect whether antiserafrom H1 (Brisbane) vaccination can inhibit NIBRG-121 (Pandemic 2009A(H1N1) vaccine strain) virus's ability to agglutinate Red Blood cells.As shown in FIG. 15A, antisera from vaccination with mono-glycosylatedHA (HA_(mg)) from H1 (Brisbane) demonstrated better ability to inhibitthe NIBRG-121 (H1N1/2009) virus's ability to agglutinate Red Blood Cellsthan fully glycosylated HA (HA_(fg)) and unglycosylated HA (HA_(ug)).

A microneutralization assay was used to detect whether antisera from H1(Brisbane) vaccination can neutralize NIBRG-121 (Pandemic 2009 A(H1N1)vaccine strain) virus's ability to infect MDCK cells. As shown in FIG.15B, mono-glycosylated HA (HA_(mg)) from H1 (Brisbane) demonstratedbetter ability to neutralize NIBRG-121 (H1N1/2009) virus's ability toinfect MDCK cells than fully glycosylated HA (HA_(fg)) andunglycosylated HA (HA_(mg)).

A virus challenge experiment was conducted to demonstrate vaccinationwith mono-glycosylated HA from H1 (Brisbane) can protect NIBRG-121(Pandemic 2009 A(H1N1) vaccine strain) virus challenge. As shown in FIG.15C, mono-glycosylated HA (Brisbane) as a vaccine protects BALB/c micefrom NIBRG-121 (Pandemic 2009 A(H1N1) vaccine strain) virus challenge.In contrast, fully glycosylated HA, which is present in traditional fluvaccines made from inactivated viruses, reveals no cross-protectiveability against H1N1 (Pandemic 2009 A(H1N1) vaccine strain) virusinfection.

FIGS. 16A-16C show inhibition of WSN (H1N1) 1933 by antisera generatedusing mono-glycosylated H1 (Brisbane) HA as antigen. FIG. 16A showsinhibition of the ability of the WSN (H1N1) 1933 virus to agglutinatered blood cells. FIG. 16B shows inhibition of the ability of the WSN(H1N1) 1933 virus to infect MDCK cells. FIG. 16C shows protection ofBALB/c mice from infection by WSN (H1N1) 1933 influenza virus. Theantisera used was mice immunized with Brisbane HA proteins (5 μg) andthe virus used for challenge was WSN (H1N1) 1933 (100×LD₅₀).

As shown in FIG. 16A, antisera from vaccination with mono-glycosylatedHA (HA_(mg)) from H1 (Brisbane) demonstrated better ability to inhibitthe WSN (H1N1) 1933 virus' ability to agglutinate Red Blood Cells thanfully glycosylated HA (HA_(fg)) and unglycosylated HA (HA_(ug)). Asshown in FIG. 16B, mono-glycosylated HA (HA_(mg)) from H1 (Brisbane)demonstrated better ability to neutralize WSN (H1N1) 1933 virus' abilityto infect MDCK cells than fully glycosylated HA (HA_(fg)) andunglycosylated HA (HA_(ug)). As shown in FIG. 16C, mono-glycosylated HA(Brisbane) as a vaccine protects BALB/c mice from WSN (H1N1) 1933 viruschallenge. In contrast, fully glycosylated HA, which is present intraditional flu vaccines made from inactivated viruses, reveals nocross-protective ability against WSN (H1N1) 1933 virus infection.

FIGS. 17A-17C show inhibition of A/Puerto Rico/8/34 (H1N1): PR8 byantisera generated using mono-glycosylated H1 (Brisbane) HA as antigen.FIG. 17A shows inhibition of the ability of the PR8 virus to agglutinatered blood cells. FIG. 17B shows inhibition of the ability of the PR8virus to infect MDCK cells. FIG. 17C shows protection of BALB/c micefrom infection by PR8 influenza virus. The antisera used was miceimmunized with Brisbane HA proteins (5 μg) and the virus used forchallenge was PR8 (100×LD₅₀)

As shown in FIG. 17A, antisera from vaccination with mono-glycosylatedHA (HA_(mg)) from H1 (Brisbane) demonstrated better ability to inhibitthe PR8 virus' ability to agglutinate Red Blood Cells than fullyglycosylated HA (HA_(fg)) and unglycosylated HA (HA_(ug)). As shown inFIG. 17B, mono-glycosylated HA (HA_(mg)) from H1 (Brisbane) demonstratedbetter ability to neutralize PR8 virus' ability to infect MDCK cellsthan fully glycosylated HA (HA_(fg)) and unglycosylated HA (HA_(ug)). Asshown in FIG. 17C, mono-glycosylated HA (Brisbane) as a vaccine protectsBALB/c mice from PR8virus challenge. In contrast, fully glycosylated HA,which is present in traditional flu vaccines made from inactivatedviruses, reveals no cross-protective ability against PR8 virusinfection.

(B) Vaccination with New H1 (Pandemic 2009 a(H1N1) Vaccine Strain)Mono-Glycosylated HA Protein.

The influenza H1 (Pandemic 2009 A(H1N1) vaccine strain) HA codingsequence was isolated and modified for expression as described inExample 1. Table 3 shows the sequence of the modified Pandemic 2009A(H1N1) vaccine strain H1/HA del-TM-FH6 where the signal peptidesequence is underlined and in bold, the thrombin cleavage site is initalics, the bacteriophage T4 fibritin foldon trimerization sequence andthe His-tag is underlined, and the linker sequence is in bold and isunderlined.

TABLE 3Influenza H1 (Pandemic 2009 A(H1N1) vaccine strain) hemagglutininsequence Amino acid sequence of influenza Pandemic 2009 A(H1N1) vaccinestrain HA del-TM-FH6 MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLL 50 EDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETP 100SSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVT 150AACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPS 200TSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTL 250VEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPK 300GAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAI 350AGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVI 400EKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENER 450TLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGT 500YDYPKYSEEAKLNREEIDGV DIRS LVPRGS PGSGYIPEAPRDGQAYVRKD 550GEWVLLSTFLGHHHHHH (SEQ ID NO: 6)Nucleotide sequence of influenza Pandemic 2009 A(H1N1) vaccinestrain HA del-TM-FH6    1ATGGCGCGCC GCTAGCATGA AGGCCATCCT GGTTGTGCTG CTGTACACCT   51TCGCTACCGC CAACGCCGAT ACCCTGTGCA TCGGCTACCA CGCCAACAAC  101AGCACCGACA CCGTGGATAC CGTGCTGGAA AAGAACGTGA CCGTGACCCA  151CAGCGTGAAC CTGGTGGAAG ATAAGCACAA CGGCAAGCTG TGCAAGCTGA  201GAGGCGTGGC CCCTCTGCAC CTGGGCAAGT GCAATATCGC CGGCTGGATC  251CTGGGCAACC CCGAGTGCGA GAGCCTGAGC ACCGCCAGCA GCTGGTCCTA  301CATCGTGGAG ACACCCAGCA GCGACAATGG CACCTGTTAC CCCGGCGACT  351TCATCGACTA CGAGGAACTG CGGGAGCAGC TGAGCAGCGT GTCCAGCTTC  401GAGCGGTTCG AGATCTTCCC CAAGACCAGC TCTTGGCCCA ACCACGACAG  451CAACAAGGGC GTGACCGCCG CCTGTCCTCA CGCTGGCGCC AAGAGCTTCT  501ACAAGAACCT GATCTGGCTG GTCAAGAAGG GCAACAGCTA CCCCAAACTG  551AGCAAGAGCT ACATCAACGA CAAGGGCAAA GAAGTGCTGG TGCTGTGGGG  601CATCCACCAC CCTAGCACCA GCGCCGACCA GCAGAGCCTG TACCAGAACG  651CCGACGCCTA CGTGTTCGTG GGCAGCAGCC GGTACAGCAA GAAGTTCAAG  701CCCGAGATCG CCATCAGACC CAAAGTGCGG GACCAAGAGG GCCGGATGAA  751CTACTACTGG ACCCTGGTGG AGCCCGGCGA CAAGATCACC TTCGAGGCCA  801CCGGCAATCT GGTCGTGCCC AGATACGCCT TCGCCATGGA AAGAAACGCC  851GGCAGCGGCA TCATCATCAG CGACACCCCC GTGCACGACT GCAACACCAC  901CTGTCAGACC CCCAAAGGCG CCATCAACAC CAGCCTGCCC TTCCAGAACA  951TCCACCCCAT CACCATCGGC AAGTGCCCTA AGTACGTGAA GTCTACCAAG 1001CTGAGGCTGG CCACAGGCCT GCGGAACATC CCCAGCATCC AGAGCAGAGG 1051CCTGTTTGGC GCCATTGCCG GCTTTATCGA GGGCGGCTGG ACCGGAATGG 1101TGGATGGATG GTATGGCTAC CACCACCAGA ATGAGCAGGG AAGCGGCTAC 1151GCCGCCGACC TGAAGTCCAC ACAGAACGCC ATCGACGAGA TCACCAACAA 1201AGTGAACTCA GTGATCGAGA AGATGAACAC CCAGTTCACC GCCGTGGGCA 1251AAGAATTCAA CCACCTGGAA AAGCGGATCG AGAACCTGAA CAAGAAGGTG 1301GACGACGGCT TCCTGGACAT CTGGACCTAC AACGCCGAGC TGCTCGTGCT 1351GCTGGAAAAC GAGCGGACCC TGGACTACCA CGACTCCAAC GTGAAGAATC 1401TGTACGAGAA AGTTCGCTCC CAGCTGAAGA ACAACGCCAA AGAGATCGGC 1451AACGGCTGCT TCGAGTTCTA CCACAAGTGC GACAACACCT GTATGGAAAG 1501CGTGAAGAAC GGCACCTACG ACTACCCCAA GTACAGCGAG GAAGCCAAGC 1551TGAACCGGGA AGAGATCGAC GGCGTGGATA TCAGATCTCT GGTGCCAAGA 1601GGATCTCCAG GATCTGGATA CATCCCAGAG GCTCCAAGAG ATGGACAAGC 1651TTACGTGAGA AAGGACGGAG AGTGGGTGCT GCTGTCTACT TTCCTGGGAC 1701ACCACCACCA CCACCACTAA (SEQ ID NO: 7)

A hemagglutination inhibition assay was used to detect whether antiserafrom H1 (Pandemic 2009 A(H1N1) vaccine strain) vaccination can inhibitWSN (H1N1) virus's ability to agglutinate Red Blood cells. As shown inFIG. 18A, antisera from vaccination with mono-glycosylated HA (HA_(mg))from H1 (Pandemic 2009 A(H1N1) vaccine strain) demonstrated betterability to inhibit the WSN (H1N1) virus's ability to agglutinate RedBlood Cells than fully glycosylated HA (HA_(fg)) and unglycosylated HA(HA_(ug)).

A microneutralization assay was used to detect whether antisera from H1(Pandemic 2009 A(H1N1) vaccine strain) vaccination can neutralize WSN(H1N1) virus's ability to infect MDCK cells. As shown in FIG. 18B,mono-glycosylated HA (HA_(mg)) from H1 (Pandemic 2009 A(H1N1) vaccinestrain) demonstrated better ability to neutralize WSN (H1N1) virus'sability to infect MDCK cells than fully glycosylated HA (HA_(fg)) andunglycosylated HA (HA_(ug)).

A virus challenge experiment is used to demonstrate vaccination withmono-glycosylated HA from H1 (Pandemic 2009 A(H1N1) vaccine strain) canprotect from WSN (H1N1) 1933 or A/Puerto Rico/8/34 (H1N1): PR8 viruschallenge. Mono-glycosylated HA (Pandemic 2009 A(H1N1) vaccine strain)as a vaccine protects BALB/c mice from WSN (H1N1) or PR8 viruschallenge. In contrast, fully glycosylated HA, which is present intraditional flu vaccines made from inactivated viruses, reveals nocross-protective ability against WSN (H1N1) or PR8 virus infection.

Partially glycosylated (e.g., mono-glycosylated) HA from other strainsof influenza virus also can be used to formulate potent vaccines activein preventing or reducing infections by one or more strains or subtypesof influenza virus. The influenza HA coding sequence from any number canbe isolated and modified for expression as described in Example 1. TheHA is then cloned and expressed in an eukaryotic expression system andthen subjected to deglycosylation to retain one to three glycosylations(preferably mono-glycosylated) at a glycosylation site.

Table 4 shows the consensus sequence of the modified H1 A del-TM-FH6where the signal peptide sequence is underlined and in bold, thethrombin cleavage site is in italics, the bacteriophage T4 fibritinfoldon trimerization sequence and the His-tag is underlined, and thelinker sequence is in bold and is underlined.

TABLE 4 Consensus H1 A del-TM-FH6 hemagglutinin sequenceConsensus amino acid sequence of influenza H1 A del-TM-FM hemagglutininMKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLL  50EDSHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVETP 100NPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTKGVS 150ASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPP 200NIGDQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTL 250LEPGDTIIFEANGNLIAPRYAFALSRGFGSGIITSNAPMDECDAKCQTPQ 300GAINSSLPFQNVHPVTIGECPKYVRSTKLRMVTGLRNIPSIQSRGLFGAI 350AGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVI 400EKMNTQFTAVGKEFNKLERRMENLNKKVDDGFLDIWTYNAELLVLLENER 450TLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGT 500YDYPKYSEESKLNREKIDGV DIRS LVPRGS PGSGYIPEAPRDGQAYVRKD 550GEWVLLSTFLGHHHHHH (SEQ ID NO: 8)Nucleotide sequence of influenza H1 A del-TM-FH6 hemagglutinin    1ATGAAGGTGA AACTGCTGGT GCTGCTGTGC ACCTTCACCG CCACCTACGC   51CGACACCATC TGCATCGGCT ACCACGCCAA CAACAGCACC GACACCGTGG  101ATACCGTGCT GGAAAAGAAC GTGACCGTGA CCCACAGCGT GAACCTGCTG  151GAAGATAGCC ACAACGGCAA GCTGTGCCTG CTGAAGGGCA TTGCCCCCCT  201GCAGCTGGGC AACTGTAGCG TGGCCGGCTG GATTCTGGGC AACCCCGAGT  251GCGAGCTGCT GATCAGCAAA GAGTCCTGGT CCTACATCGT GGAGACACCC  301AACCCCGAGA ACGGCACCTG TTACCCCGGC TACTTCGCCG ACTACGAGGA  351ACTCACACAG CAGCTGTCCT CTGTCTCCAG CTTCGAGCGG TTCGAGATCT  401TCCCCAAAGA GAGCAGCTGG CCCAACCACA CCGTGACAAA GGGCGTGAGC  451GCCAGCTGCT CCCACAATGG CAAGAGCAGC TTCTACCGGA ACCTGCTGTG  501GCTGACCGGC AAGAACGGCC TGTACCCCAA CCTGAGCAAG AGCTATGCCA  551ACAACAAAGA GAAAGAGGTC CTCGTCCTCT GGGGCGTGCA CCACCCCCCC  601AACATCGGCG ACCAGCGGGC CCTGTACCAC ACCGAGAACG CCTACGTGTC  651CGTGGTGTCC AGCCACTACA GCAGACGGTT CACCCCCGAG ATCGCCAAGA  701GGCCCAAAGT GCGGGACCAG GAAGGCCGGA TCAACTACTA CTGGACCCTG  751CTGGAACCCG GCGACACCAT CATCTTCGAG GCCAACGGCA ACCTGATCGC  801CCCCAGATAC GCCTTTGCCC TGAGCAGAGG CTTCGGCAGC GGCATCATCA  851CCAGCAACGC CCCCATGGAC GAGTGCGACG CCAAGTGTCA GACCCCCCAG  901GGCGCCATCA ACAGCAGCCT GCCCTTCCAG AACGTGCACC CCGTGACCAT  951CGGCGAGTGC CCTAAGTACG TGCGGAGCAC CAAGCTGAGA ATGGTGACCG 1001GCCTGCGGAA CATCCCCAGC ATCCAGAGCA GAGGCCTGTT TGGCGCCATT 1051GCCGGCTTTA TCGAGGGCGG CTGGACCGGA ATGGTGGACG GGTGGTACGG 1101CTACCACCAC CAGAATGAGC AGGGCAGCGG CTACGCCGCC GATCAGAAGT 1151CCACCCAGAA CGCTATCAAC GGCATCACCA ACAAAGTGAA CAGCGTGATC 1201GAGAAGATGA ACACCCAGTT CACCGCCGTG GGCAAAGAGT TCAACAAGCT 1251GGAACGGCGG ATGGAAAACC TGAACAAGAA GGTGGACGAC GGCTTCCTGG 1301ACATCTGGAC CTACAACGCC GAGCTGCTGG TCCTGCTGGA AAACGAGCGG 1351ACCCTGGACT TCCACGACAG CAACGTGAAG AACCTGTACG AGAAAGTGAA 1401GTCCCAGCTG AAGAACAACG CCAAAGAGAT CGGCAACGGC TGCTTCGAGT 1451TCTACCACAA GTGCAACGAC GAGTUCATGG AAAGCGTGAA GAACGGCACA 1501TACGACTACC CCAAGTACAG CGAGGAAAGC AAGCTGAACC GGGAGAAGAT 1551CGACGGCGTG GATATCAGAT CTCTGGTGCC AAGAGGATCT CCAGGATCTG 1601GATACATCCC AGAGGCTCCA AGAGATGGAC AAGCTTACGT GAGAAAGGAC 1651GGAGAGTGGG TGCTGCTGTC TACTTTCCTG GGACACCACC ACCACCACCA 1701CTAA (SEQ ID NO: 9)

Table 5 shows the consensus sequence of the modified H1-C del-TM-FH6where the signal peptide sequence is underlined and in bold, thethrombin cleavage site is in italics, the bacteriophage T4 fibritinfoldon trimerization sequence and the His-tag is underlined, and thelinker sequence is in bold and is underlined.

TABLE 5 Consensus H1-C del-TM-FH6 hemagglutinin sequenceConsensus amino acid sequence of influenza H1-C del-TM-FH6 hemagglutininMKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLL  50EDSHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESESYIVETP 100NPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKESSWPNHDTVTGVS 150ASCSHNGESSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPP 200NIGDQKALYHTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTL 250LEPGDTIIFEANGNLIAPRYAFALSRGFGSGIINSNAPMDKCDAKCQTPQ 300GAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAI 350AGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVI 400EKMNTQFTAVGKEFNKLERRMENLNKKVDDGFLDIWTYNAELLVLLENER 450TLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGT 500YDYPKYSEESKLNREKIDGV DIRS LVPRGS PGSGYIPEAPRDGQAYVRKD 550GEWVLLSTFLGHHHHHH (SEQ ID NO: 10)Nucleotide sequence of influenza H1-C del-TM-FH6 hemagglutnin    1ATGAAGGTGA AACTGCTGGT GCTGCTGTGC ACCTTCACCG CCACCTACGC   51CGACACCATC TGCATCGGCT ACCACGCCAA CAACAGCACC GACACCGTGG  101ATACCGTGCT GGAAAAGAAC GTGACCGTGA CCCACAGCGT GAACCTGCTG  151GAAGATAGCC ACAACGGCAA GCTGTGCCTG CTGAAGGGCA TTGCCCCCCT  201GCAGCTGGGC AACTGTAGCG TGGCCGGCTG GATTCTGGGC AACCCCGAGT  251GCGAGCTGCT GATCTCCAAA GAGTCCTGGT CTTACATCGT GGAGACACCC  301AACCCCGAGA ACGGCACCTG TTACCCCGGC CACTTCGCCG ACTACGAGGA  351ACTGCGGGAG CAGCTGAGCA GCGTGTCCAG CTTCGAGCGG TTCGAGATCT  401TCCCCAAAGA GAGCAGCTGG CCCAACCACG ATACCGTGAC CGGCGTGAGC  451GCCAGCTGTT CCCACAACGG CGAGAGCAGC TTCTACCGGA ACCTGCTGTG  501GCTGACCGGC AAGAACGGCC TGTACCCCAA CCTGAGCAAG AGCTATGCCA  551ACAACAAAGA GAAGGAAGTC CTGGTCCTCT GGGGCGTGCA CCACCCCCCC  601AACATCGGCG ACCAGAAGGC CCTGTACCAC ACCGAGAACG CCTACGTGTC  651CGTGGTGTCC AGCCACTACA GCCGGAAGTT CACCCCCGAG ATCGCCAAGA  701GGCCCAAAGT GCGGGACCAG GAAGGCCGGA TCAACTACTA CTGGACCCTG  751CTGGAACCCG GCGACACCAT CATCTTCGAG GCCAACGGCA ACCTGATCGC  801CCCCAGATAC GCCTTTGCCC TGAGCAGAGG CTTCGGCAGC GGCATCATCA  851ACAGCAACGC CCCCATGGAC AAGTGCGACG CCAAGTGCCA GACACCCCAG  901GGCGCCATCA ACAGCTCCCT GCCCTTCCAG AACGTGCACC CCGTGACCAT  951CGGCGAGTGC CCTAAGTACG TGCGGAGCGC CAAGCTGAGA ATGGTGACCG 1001GCCTGCGGAA CATCCCCAGC ATCCAGAGCA GAGGCCTGTT TGGCGCCATT 1051GCCGGCTTTA TCGAGGGCGG CTGGACCGGA ATGGTGGACG GGTGGTACGG 1101CTACCACCAC CAGAATGAGC AGGGCAGCGG CTACGCCGCC GATCAGAAGT 1151CCACCCAGAA CGCCATCAAC GGCATCACCA ACAAAGTGAA CAGCGTGATC 1201GAGAAGATGA ACACCCAGTT CACCGCCGTG GGCAAAGAGT TCAACAAGCT 1251GGAACGGCGG ATGGAAAACC TGAACAAGAA GGTGGACGAC GGCTTCCTGG 1301ACATCTGGAC CTACAACGCC GAGCTGCTGG TGCTGCTGGA AAACGAGCGG 1351ACCCTGGACT TCCACGACAG CAACGTGAAG AACCTGTACG AGAAAGTGAA 1401GTCCCAGCTG AAGAACAACG CCAAAGAGAT CGGCAACGGC TGCTTCGAGT 1451TCTACCACAA GTGCAACGAC GAGTGCATGG AAAGCGTGAA GAACGGCACA 1501TACGACTACC CCAAGTACAG CGAGGAAAGC AAGCTGAACC GGGAGAAGAT 1551CGACGGCGTG GATATCAGAT CTCTGGTGCC AAGAGGATCT CCAGGATCTG 1601GATACATCCC AGAGGCTCCA AGAGATGGAC AAGCTTACGT GAGAAAGGAC 1651GGAGAGTGGG TGCTGCTGTC TACTTTCCTG GGACACCACC ACCACCACCA 1701CTAA (SEQ ID NO: 11)

Vaccines generated from the deglycosylated HA peptides of the instantdisclosure exhibit antiviral activity against respiratory viruses,including respiratory syncytial virus (RSV) and various types ofinfluenza, such as influenza A and influenza B. Advantageously, theantiviral peptides of the present disclosure exhibit antiviral activityagainst numerous strains of influenza, including seasonal, avian (e.g.,H5N1 strains), and swine influenzas. Illnesses resulting from infectionsby these viruses can also be prevented or treated according to some ofthe disclosed methods.

(C) Glycosylation Sites on the H1/HA Protein.

The influenza H1 HA molecules have four distinct antigenic sites: Sa,Sb, Ca, and Cb (Luoh S M, et al. (1992) J Virol 66:1066-1073). Thesesites consist of the most variable amino acids in the HA molecule of theseasonal human H1N1 viruses that have been subjected toantibody-mediated immune pressure since its emergence in 1918.

Using hemagglutination inhibition (HI) assays and vaccination/challengestudies, it was demonstrated that the 2009 pandemic H1N1 virus isantigenically similar to human H1N1 viruses that circulated from1918-1943 and to classical swine H1N1 viruses. Antibodies against1918-like or classical swine H1N1 vaccines were found to completelyprotect C57B/6 mice from lethal challenge with the influenzaA/Netherlands/602/2009 virus isolate. Passive immunization withcross-reactive monoclonal antibodies (mAbs) raised against either 1918or A/California/04/2009 HA proteins were found to offer full protectionfrom death. Analysis of mAh antibody escape mutants, generated byselection of 2009 H1N1 virus with these mAbs, indicate that antigenicsite Sa is one of the conserved cross-protective epitopes. (ManicassamyB., et al. PLoS Pathogens January 2010|Volume 6|Issue 1|e1000745).

By homology modeling of the HA structure, it has been shown that HAs of2009 H1N1 and the 1918 pandemic virus share a significant number ofamino acid residues in known antigenic sites, suggesting the existenceof common epitopes for neutralizing antibodies cross-reactive to bothHAs. (Igarashi M. et al., PLoS ONE January 2010, Volume 5, Issue 1,e8553). A potential glycosylation site exists at the Asn177 residue onHA, which is within the antigenically conserved Sa region. (See FIG. 19). Proteins carrying a mutation at the Asn177 glycosylation site withinthe HA of Brisbane H1 are used to immunize mice. Cross-protectionagainst NIBRG-121 is measured.

Antisera from vaccination with mono-glycosylated HA (HA_(mg)) carrying amutation at Asn177 from H1 (Brisbane) demonstrates better ability toinhibit the NIBRG-121 virus' ability to agglutinate Red Blood Cells thanfully glycosylated HA (HA_(fg)) carrying a mutation at Asn177 andunglycosylated HA (HA_(ug)) carrying a mutation at Asn177.Mono-glycosylated HA (HA_(mg)) carrying a mutation at Asn177 from H1(Brisbane) demonstrates better ability to neutralize NIBRG-121 virus'ability to infect MDCK cells than fully glycosylated HA (HA_(fg))carrying a mutation at Asn177 and unglycosylated HA (HA_(ug)) carrying amutation at Asn177. Mono-glycosylated HA (Brisbane) as a vaccineprotects BALB/c mice from NIBRG-121 virus challenge. In contrast, fullyglycosylated HA carrying a mutation at Asn177 reveals little or nocross-protective ability against NIBRG-121 virus infection.

As used herein, “therapeutic activity” or “activity” may refer to anactivity whose effect is consistent with a desirable therapeutic outcomein humans, or to desired effects in non-human mammals or in otherspecies or organisms. Therapeutic activity may be measured in vivo or invitro. For example, a desirable effect may be assayed in cell culture.

The “antiviral activity” of a vaccine according the present disclosuredenotes the ability of the vaccine to generate an immune response in asubject to whom the vaccine is administered wherein the immune responseis sufficient to prevent or treat or ameliorate full blown viralinfection and/or symptoms associated with infection by a virus, such asan influenza virus. Advantageously, vaccines generated from thedeglycosylated HA peptides of the instant disclosure may demonstratesignificant antiviral activity against influenza virus. As used herein,“significant antiviral activity” can be measured by the ability of thevaccine to inhibit viral hemagglutination by at least about 50%, ascompared to mock treated samples of virus. In certain embodiments, theantiviral peptide inhibits viral hemagglutination by at least about 60%,more preferably by at least about 70%, more preferably by at least about80%, more preferably by at least about 90%, and more preferably by atleast about 95%, as compared to mock treated samples of virus.

Methods for demonstrating the inhibitory effect of antiviralcompositions on viral replication are well known in the art. Thetherapeutic efficacy of the vaccines of the present invention asantiviral agents can be demonstrated in laboratory animals, for example,by using a murine model. (See e.g., Jones, et al., J. Virol, 2006, Vol.80, No. 24, pp. 11960-11967). Additionally, the therapeutic effect ofthe pharmacologically active peptides of the present invention can beshown in humans via techniques known in the art.

The neutralizing antibodies of the present invention can be additionallyused as a tool for epitope mapping of antigenic determinants ofinfluenza A virus, and are useful in vaccine development. Indeed, asshown in the Examples below, the inventors herein have identifiedseveral broadly reactive neutralizing antibodies that can be used asguides for vaccine design.

Thus, the neutralizing antibodies of the present invention can be usedto select peptides or polypeptides that functionally mimic theneutralization epitopes to which the antibodies bind, which, in turn,can be developed into vaccines against influenza A virus infection. Inone embodiment, the present invention provides a vaccine effectiveagainst an influenza A virus comprising a peptide or polypeptide thatfunctionally mimics a neutralization epitope bound by an antibodydescribed herein. In one embodiment, the vaccine comprises a peptide orpolypeptide functionally mimicking a neutralization epi tope bound by anantibody that hinds a hemagglutinin (HA) antigen. In another embodiment,the vaccine may be synthetic. In other embodiments, the vaccine maycomprise (i) an attenuated influenza A virus, or a part thereof; or (ii)a killed influenza A virus, or part thereof. In one other embodiment,the vaccine comprises a peptide or polypeptide functionally mimicking aneutralization epitope bound by an antibody that binds a hemagglutinin(HA) antigen. The HA antigen may be an H5 subtype or an H1 subtype. Insome embodiments, the HA antigen is displayed on the surface ofinfluenza A virus.

Influenza Virus Vaccines

The N-glycosylation site sequences of influenza H5 HA are highlyconserved. The H5 HA has 15 total N-glycosylation sites having theprototypic sequence N-X-(S/T). Each monomer has 5 N-glycosylation sitesat positions N27, N39, N170, N181 and N500. Host receptor binding isaffected by glycans on HA structure. H5 HA has glycosylation sites atpositions 39, 127, 170, 181, and 500.

The vaccines of the invention can be generated using partiallyglycosylated versions of any surface protein of influenza virus,including HA, NA and M2. The influenza A virus neuraminidase (NA)proteins are displayed on their surface. The influenza A virus M2protein is an integral membrane protein of 97 amino acids that isexpressed at the surface of infected cells with an extracellularN-terminal domain of 18 to 23 amino acid residues, an internalhydrophobic domain of approximately 19 residues, and a C-terminalcytoplasmic domain of 54 residues. (Zebedee S L, et al. J. Virol. 1988August; 62(8):2762-2772).

Further, the partially glycosylated influenza proteins may be generatedby altering the glycosylation pattern at N- or O-glycosylation sites ofthe protein used as an antigen.

In another embodiment, the peptides or polypeptides of the vaccinecontain antigenic determinants that raise influenza A virus neutralizingantibodies.

In a more general aspect, the neutralizing molecules, including but notlimited to antibodies, are useful to prevent or treat viral infections.Thus, the neutralizing molecules of the present invention are useful inimmunotherapy, such as passive immunization using one or more suchmolecules, and in the development of vaccines directed at the viralantigenic target(s).

The present provides vaccine compositions for the prevention andtreatment of infections caused by the avian influenza neutralized virus.While it has been known for over 80 years that passive administration ofimmune sera can prevent infection Luke, T. C. et al., Kilbane E M,Jackson J L, & Hoffman S L (2006) Ann Intern Med 145, 599-609), morerecent studies with monoclonal antibodies also offer encouragement(Hanson, B. J. et al. (2006) Respir Res 7, 126; Huang, C. C. et al.(2004) Proc. Nat. Acad. Sci. 101, 2706-2711; Simmons C. P. et al. (2007)PLoS Med 4, e178). For example, Hanson et al. showed that a monoclonalantibody to H5N1 virus was protective against lethal infection, evenwhen given three days post inoculation in mice (Hanson, B. J. et al.(2006) Respir Res 7, 126).

Given the possibility of a catastrophic epidemic, it has been suggestedthat governments should maintain stocks of neutralizing antibodies suchas those reported here. The facts that antibodies are fully human andhave been isolated from individuals who successfully combated viralinfection may offer advantages. However, even if such antibodies arestockpiled, if the gene encoding the epitope to which the antibody bindswere to mutate, then the antibody might be less effective. Also, thereis some evidence that cellular immunity enhances clearance of the virus.Nevertheless, if the only effect of passive immunization was to lessenthe severity of infection, thereby giving the necessary time for otherimmune effectors to operate, it could be of critical importance forlessening mortality in patients with weakened immune, cardiovascular,and respiratory systems and in the elderly. Passive immunization mightprevent cytokine storm against rapidly proliferating viruses, asoccurred even in healthy young adults during the 1918 influenzaoutbreak.

Respiratory Syncytial Virus (RSV) Vaccines

Human respiratory syncytial virus (RSV) is a virus that causesrespiratory tract infections. It is the major cause of lower respiratorytract infection and hospital visits during infancy and childhood. RSV isan enveloped RNA virus of the family Paramyxoviridae and of the genusPneumovirus. There is no vaccine.

The RSV virion comprises three surface glycoproteins, three surfaceglycoproteins, F, G and SH (small hydrophobic). F proteins on thesurface of the virus cause the cell membranes on nearby cells to merge,forming syncytia. F (fusion) and G (attachment) glycoproteins arerequired for viral entry into cells and they also determine the antibodyresponse. The structure and composition of RSV has been elucidated andis described in detail in the textbook “Fields Virology”, ed. Knipe, D.M. et al., Lippincott Williams & Wilkins, NY (2001), in particular,Chapter 45, pp. 1443-1485, “Respiratory Syncytial Virus” by Collins, P.,Chanock, R. M. and Murphy, B. R.

RSV G protein, which is 33 kDa unglycosylated, runs at approximately 90kDa when fully glycosylated (both N- and O-linked glycosylations). F andG proteins exist as a protein complex on the surface of RSV-infectedcells. (Low K-W et al. Biochem. Biophys. Res. Comm 366(2) 2008,308-313).

Table 6 indicates the sequence of the RSV glycoprotein G with potentialN-glycosylation sites underlined.

TABLE 6 RSV glycoprotein G polypeptide sequence.MSKNKDQRTT KTLEKTWDTL NHLLFISSCL YKLNLKSIAQ ITLSILAMII STSLIIAAII  60FIASANHKVT LTTAIIQDAT SQIK N TTPTY LTQNPQLGIS FS N LSETTSQ TTTILASTTP120 SVKSTLQSTT VKTKNTTTTK IQPSKPTTKQ RQNKPPNKPN NDFHFEVFNF VPCSICSNNP180 TCWAICKRIP NKKPGKKTTT KPTKKPTIKT TKKDLKPQTT KPKEVPTTKP TEKPTI N TTK240 TNIRTTLLT N   N TTNNPEHTS QKGTLHSTSS DGNPSPSQVY TTSEYLSQPP SPS NTTNQ 298 (SEQ ID NO: 12)

Partially deglycosylated RSV glycoproteins F and U could be useful asmore effective RSV vaccines.

Flavivirus Vaccines

Flavivirus is a genus of the family Flaviviridae. Flaviviruses aresmall, enveloped RNA viruses that use arthropods such as mosquitoes fortransmission to their vertebrate hosts, and include Yellow fever virus(YFV), West Nile virus (WNV), Tick-borne encephalitis virus, Japaneseencephalitis virus (JE) and Dengue virus 2 viruses (Weaver S C, BarrettA D Nat. Rev. Microbiol. 2 789-801 2004). Flaviviruses consist of threestructural proteins: the core nucleocapsid protein C, and the envelopeglycoproteins M and E. Glycoprotein E is a class 11 viral fusion proteinthat mediates both receptor binding and fusion. Class II viral fusionproteins are found in flaviviruses and alphaviruses.

Glycoprotein E is comprised of three domains: domain I (dimerisationdomain) is an 8-stranded beta barrel, domain II (central domain) is anelongated domain composed of twelve beta strands and two alpha helices,and domain III (immunoglobulin-like domain) is an IgC-like module withten beta strands. Domains I and II are intertwined.

The 495 AA glycoprotein E dimers on the viral surface re-clusterirreversibly into fusion-competent trimers upon exposure to low pH, asfound in the acidic environment of the endosome. The formation oftrimers results in a conformational change that results in the exposureof a fusion peptide loop at the tip of domain II, which is required inthe fusion step to drive the cellular and viral membranes together byinserting into the membrane (Modis Y et al., Proc. Natl. Acad. Sci.U.S.A. 100 6986-91 2003).

Dengue virus envelope protein (E) contains two major N-linkedglycosylation sites, at Asn-67 and Asn-153. The glycosylation site atposition 153 is conserved in most flaviviruses, while the site atposition 67 is thought to be unique for dengue viruses. N-linkedoligosaccharide side chains on flavivirus E proteins have beenassociated with viral morphogenesis, infectivity, and tropism. Dengueviruses lacking N-glycosylation at position 67 show reduced infection ofhuman cells. (Mondotte J A, et al., J. Virol. 81(3):7136-7148 (2007).

Table 7 indicates the sequence of the Dengue virus glycoprotein E withpotential N-glycosylation sites at N-67 and N-153 underlined.

TABLE 7 Dengue virus glycoprotein E polypeptide sequence.MRCVGIGNRD FVEGLSGATW VDVVLEHGSC VTTMAKNKPT LDIELLKTEV TNPAVLRKLC  60IEAKIS N TTT DSRCPTQGEA TLVEEQDANF VCRRTFVDRG WGNGCGLFGK GSLLTCAKFK 120CVTKLEGKIV QYENLKYSVI VTVHTGDQHQ VG N ETTEHGT IATITPQAPM SEIQLTDYGA 180LTLDCSPRTG LDFNEMVLLT MKEKSWLVHK QWFLDLPLPW TSGASTSQET WNRQDLLVTF 240KTAHAKKQEV VVLGSQEGAM HTALTGATEI QTSGTTTIFA GHLKCRLKMD KLTLKGVSYV 300MCTGSFKLEK EVAETQHGTV LVQVKYEGTD APCKIPFSTQ DEKGVTQNGR LITANPIVTD 360KEKPVNIETE PPFGESYIVI GAGEKALKLS WFKKGSSIGK MFEATARGAR RMAILGDIAW 420DFGSIGGAFT SVGKLVHQVF GTAYGVLFSG VSWTNKIGIG ILLTWLGLNS RSTSLSMTCI 480AVGMVTLYLG VVVQA 495 (SEQ ID NO: 13)

Thus, partial deglycosylation of Dengue virus glycoprotein E can be usedto generate more effective and broad vaccines against flaviviruses. Theresult of partial deglycosylation of the Dengue type 3 virus E proteindimers is shown in the model depicted in FIGS. 20A-20B. FIG. 20B showsmonoglycosylated Dengue E protein.

Hepatitis C virus (HCV) is the major etiological agent of humanpost-transfusion infection and community-acquired non-A, non-Bhepatitis, infecting probably 1% of the population worldwide. HCV is amember of the Flaviviridae family, which includes the flaviviruses andthe pestiviruses (Miller R H & Purcell R H, Proc. Natl. Acad. Sci., USA87, 2057-2061 1990).

The hepatitis C virus (HCV) genome encodes two membrane-associatedenvelope glycoproteins (E1 and E2), which interact to form a noncovalentheterodimeric complex. HCV glycoproteins, E1 and E2, are heavilymodified by N-linked glycosylation. The E1 protein consists of 192 aminoacids and contains 5 to 6 N-glycosylation sites, depending on the HCVgenotype. The E2 protein consists of 363 to 370 amino acids and contains9-11 N-glycosylation sites, depending on the HCV genotype. (Maertens G.and Stuyver L. Genotypes and genetic variation of hepatitis C virus. In:The molecular medicine of viral hepatitis. Ed: Harrison T. J. andZuckerman A. J. 1997).

A recent study has revealed that upon partial deglycosylation withendoglycosidase H only four of the five potential glycosylation sites atamino acid positions 196, 209, 234, 305 and 325, respectively, of HCVglycoprotein E1 are utilized. Mutations at positions N2 (196) and N3(234) have only minor effects on the assembly of the E1E2 complex,whereas a mutation at position NI (196) and predominantly at position N4(305) dramatically reduces the efficiency of the formation ofnoncovalent E1E2 complexes. (Meunier J C. et al., J. Gen. Virol. (1999),80, 887-896.)

Table 8 indicates the sequence of the Hepatitis C virus isolate HC-J6envelope glycoprotein E1 (Okamoto, H., et al., J. Gen. Virol. 72(11),2697-2704 (1991)) with potential N-glycosylation sites at positions 196,209, 234, 305 and 325 underlined.

TABLE 8 Hepatitis C virus envelope glycoprotein E1 polypeptide sequence,AEVK N ISTGY MVTNDCT N DS ITWQLQAAVL HVPGCVPCEK VG N TSPCWIP VSPNVAVQQP252 GALTQGLRTH IDMVVMSATL CSALYVGDLC GGVMLAAQMF IVSPQHHWFV QDC N CSIYPG312 TITGHRMAWD MMM N WSPTAT MILAYAMRVP EVIIDIIGGA HWGVMFGLAY FSMQGAWAKV372 VVILLLAAGV DA 384 (SEQ ID NO: 14)

Thus, partial deglycosylation of HCV glycoproteins E1 and E2 can be usedto generate more effective and broad vaccines against HCV.

Human Immunodeficiency Virus (HIV) Vaccines

The human immunodeficiency viruses HIV-1 and HIV-2 and the relatedsimian immunodeficiency viruses (SIV) cause the destruction of CD4⁺lymphocytes in their respective hosts, resulting in the development ofacquired immunodeficiency syndrome (AIDS). The entry of HIV into hostcells is mediated by the viral envelope glycoproteins, which areorganized into oligomeric, probably trimeric spikes displayed on thesurface of the virion. These envelope complexes are anchored in theviral membrane by the gp41 transmembrane envelope glycoprotein. Thesurface of the spike is composed primarily of the exterior envelopeglycoprotein, gp120, associated by non-covalent interactions with eachsubunit of the trimeric gp41 glycoprotein complex.

The addition of asparagine (N)-linked polysaccharide chains (i.e.,glycans) to the gp120 and gp41 glycoproteins of human immunodeficiencyvirus type 1 (HIV-1) envelope is not only required for correct proteinfolding, but also may provide protection against neutralizing antibodiesas a “glycan shield.” (Wei X et al., Nature 422: 307-312, 2003). Thesurface glycoprotein (gp120) of the human immunodeficiency virus type 1(HIV-1) envelope, which represents the primary interface between thevirus and the host environment, is one of the most heavily glycosylatedproteins known to date, with nearly half of its molecular weight due tothe addition of N-linked glycans. (Allan J S, et al. Science 228:1091-1094, 1985.) The transmembrane glycoprotein (gp41) of the HIV-1envelope is also glycosylated, but to a lesser extent. The addition ofN-linked glycans is essential for HIV-1 gp120 to fold into the properconformation to bind to the CD4 receptor, and influences the binding ofalternative coreceptors CXCR4 and CCR5, the combined effects mediatingthe fusion and entry of HIV-1 into the host cell.

Because many N-linked glycans are highly conserved components of theHIV-1 envelope, they may themselves provide a promising target forneutralizing antibodies. The broadly neutralizing human monoclonalantibody 2012 binds to an epitope comprising N-linked glycans that areattached to the gp120 glycoprotein. (Trloka A et al., J Viral 70:1100-1108, 1996). Strains of HIV-1 in which N-linked glycosylation siteshave been experimentally deleted or modified can become more sensitiveto neutralization (Koch et al., 2003 Virology 313: 387-400.)

Mature gp120 contains 24 potential sites for N-glycosylation, asrecognized by the sequence Asn-Xaa-Ser/Thr. (Kornfeld and Kornfeld, AnnRev. Biochem. 54: 631-664, 1985). Table 9 indicates the 24 sites in theHIV-1 HXB2 sequence. Potential N-glycosylation sites are underlined.

TABLE 9 HIV gp120 polypeptide sequence.LWVTVYYGVP VWKEATTTLF CASDAKAYDT EVHNVWATHA CVPTDPNPQE VVLV N VTENF  60NMWKNDMVEQ MHEDIISLWD QSLKPCVKLT PLCVSLKCTD LK N DTNT N SS SGRMIMEKGE120 IK N CSF N IST SIRGKVQKEY AFFYKLDIIP ID N DTTSYKL TSC NTSVITQ ACPKVSFEPI 180 PIHYCAPAGF AILKCN N KTF  N GTGPCT NVS TVQCTHGIRP VVSTQLLL N G SLAEEEVVIR 240 SV N FTDNAKT IIVQL N TSVE I NCTRPN N NT RKRIRIQRGP GRAFVTIGKI GNMPQAHC N I 300 SRAKW NNTLK QIASKLREQF GN N KTIIFKQ SSGGDPEIVT HSFNCGGEFF YC N STQLF N S 360TWF N STWSTE GS N NTEGSDT ITLPCRIKQI INMWQKVGKA MYAPPISGQT RCSS N ITGLL420 LTRDGGNSN N  ESEIFRPGGG DMRDNWRSEL YKYKVVKIEP LGVAPTKAKR RVVQREKR478 (SEQ ID NO: 15)

In the HIV-1 transmembrane glycoprotein gp41, the conservedglycosylation sites are at Asn621, Asn630 and Asn642.

Thus, partial deglycosylation of HIV envelope protein gp120, ortransmembrane protein gp41 can be used to generate more effective andbroad vaccines against flaviviruses. The result of partialdeglycosylation of the HIV gp120 protein trimers is shown in the modeldepicted in FIGS. 21A-21B. FIG. 21B shows monoglycosylated HIV gp120protein trimers.

Methods for Making Partially Glycosylated Cell-Surface Glycoproteins

Polynucleotides of the present invention, or fragments or variantsthereof, are readily prepared by, for example, directly synthesizing thefragment by chemical means, as is commonly practiced using an automatedoligonucleotide synthesizer. Also, fragments are obtained by applicationof nucleic acid reproduction technology, such as the PCR™ technology ofU.S. Pat. No. 4,683,202, by introducing selected sequences intorecombinant vectors for recombinant production, and by other recombinantDNA techniques generally known to those of skill in the art of molecularbiology.

The invention provides vectors and host cells comprising a nucleic acidof the present invention, as well as recombinant techniques for theproduction of a polypeptide of the present invention. Vectors of theinvention include those capable of replication in any type of cell ororganism, including, e.g., plasmids, phage, cosmids, and minichromosomes. In various embodiments, vectors comprising a polynucleotideof the present invention are vectors suitable for propagation orreplication of the polynucleotide, or vectors suitable for expressing apolypeptide of the present invention. Such vectors are known in the artand commercially available.

Polynucleotides of the present invention can be synthesized, whole or inparts that are then combined, and inserted into a vector using routinemolecular and cell biology techniques, including, e.g., subcloning thepolynucleotide into a linearized vector using appropriate restrictionsites and restriction enzymes. Polynucleotides of the present inventionare amplified by polymerase chain reaction using oligonucleotide primerscomplementary to each strand of the polynucleotide. These primers alsoinclude restriction enzyme cleavage sites to facilitate subcloning intoa vector. The replicable vector components generally include, but arenot limited to, one or more of the following: a signal sequence, anorigin of replication, and one or more marker or selectable genes.

In order to express a polypeptide of the present invention, thenucleotide sequences encoding the polypeptide, or functionalequivalents, are inserted into an appropriate expression vector, i.e., avector that contains the necessary elements for the transcription andtranslation of the inserted coding sequence. Methods well known to thoseskilled in the art are used to construct expression vectors containingsequences encoding a polypeptide of interest and appropriatetranscriptional and translational control elements. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination. Such techniques are described, forexample, in Sambrook, J., et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York. N.Y.

A variety of expression vector/host systems are utilized to contain andexpress polynucleotide sequences. These include, but are not limited to,microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

A variety of promoter sequences are known for eukaryotes and any areused according to the present invention. Virtually all eukaryotic geneshave an AT-rich region located approximately 25 to 30 bases upstreamfrom the site where transcription is initiated. Another sequence found70 to 80 bases upstream from the start of transcription of many genes isa CNCAAT region where N may be any nucleotide. At the 3′ end of mosteukaryotic genes is an AATAAA sequence that may be the signal foraddition of the poly A tail to the 3′ end of the coding sequence. All ofthese sequences are suitably inserted into eukaryotic expressionvectors.

In mammalian cell systems, promoters from mammalian genes or frommammalian viruses are generally preferred. Polypeptide expression fromvectors in mammalian host cells aer controlled, for example, bypromoters obtained from the genomes of viruses such as polyoma virus,fowlpox virus, adenovirus (e.g., Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-Bvirus and most preferably Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, and from heat-shock promoters, provided such promoters arecompatible with the host cell systems. If it is necessary to generate acell line that contains multiple copies of the sequence encoding apolypeptide, vectors based on SV40 or EBV may be advantageously usedwith an appropriate selectable marker. One example of a suitableexpression vector is pcDNA-3.1 (Invitrogen, Carlsbad, Calif.), whichincludes a CMV promoter.

A number of viral-based expression systems are available for mammalianexpression of polypeptides. For example, in cases where an adenovirus isused as an expression vector, sequences encoding a polypeptide ofinterest may be ligated into an adenovirus transcription/translationcomplex consisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus that is capable of expressing thepolypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc.Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

In bacterial systems, any of a number of expression vectors are selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities are desired, vectors that direct highlevel expression of fusion proteins that are readily purified are used.Such vectors include, but are not limited to, the multifunctional E.coli cloning and expression vectors such as BLUESCRIPT (Stratagene), inwhich the sequence encoding the polypeptide of interest may be ligatedinto the vector in frame with sequences for the amino-terminal Met andthe subsequent 7 residues of β-galactosidase, so that a hybrid proteinis produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega,Madison, Wis.) are also used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems are designedto include heparin, thrombin, or factor XA protease cleavage sites sothat the cloned polypeptide of interest can be released from the GSTmoiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH are used. Examples of other suitable promoter sequencesfor use with yeast hosts include the promoters for 3-phosphoglyceratekinase or other glycolytic enzymes, such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. For reviews, see Ausubel etal. (supra) and Grant et al. (1987) Methods Enzymol. 153:516-544. Otheryeast promoters that are inducible promoters having the additionaladvantage of transcription controlled by growth conditions include thepromoter regions for alcohol dehydrogenase 2, isocytochrome C, acidphosphatase, degradative enzymes associated with nitrogen metabolism,metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Suitable vectors andpromoters for use in yeast expression are further described in EP73,657. Expression of glycosylated HCV surface proteins in yeast isdisclosed in WO 96/04385. Yeast enhancers also are advantageously usedwith yeast promoters.

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides are driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV are used alone or in combination with the omega leadersequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters are used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J., et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, e.g., Hobbs,S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology(1992) McGraw Hill, New York, N.Y.; pp.

An insect system is also used to express a polypeptide of interest. Forexample, in one such system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding thepolypeptide are cloned into a non-essential region of the virus, such asthe polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of the polypeptide-encoding sequencerenders the polyhedrin gene inactive and produce recombinant viruslacking coat protein. The recombinant viruses are then used to infect,for example, S. frugiperda cells or Trichoplusia larvae, in which thepolypeptide of interest is expressed (Engelhard, E. K. et al. (1994)Proc. Natl. Acad. Sci. 91:3224-3227).

Partial deglycosylation of the recombinant surface glycoproteins can beaccomplished by controlled use of combinations of various glycosidases,such as treating with neuraminidase to remove sialic acid, withalpha-1-mannosidase (Sigma) to cleave external mannose residues, or withendo F-N glycanase ((Boehringer Mannheim Biochemicals, Mannheim,Germany), which efficiently cleaves both N-linked high-mannose andcomplex glycans.

The HA peptides of the present invention also can be synthesized byprocesses which incorporate methods commonly used in peptide synthesissuch as classical solution coupling of amino acid residues and/orpeptide fragments, and, if desired, solid phase techniques. Any methodfor peptide synthesis well known in the art may be used, for example,Schroeder and Lubke, in “The Peptides”, Vol. 1, Academic Press, NewYork, N.Y., pp. 2-128 (1965); “The Peptides: Analysis, Synthesis,Biology”, (E. Gross et al., Eds.), Academic Press, New York, N.Y., Vol.1-8, (1979-1987); Stewart and Young, in “Solid Phase Peptide Synthesis”,2nd Ed., Pierce Chem. Co., Rockford, Ill. (1984); Wild et al., Proc.Natl. Acad. Sci. USA, 89: 10537 (1992); and Rimsky et al., J Virol, 72:986 (1998); Chan & White in “Fmoc Solid Phase Peptide Synthesis: APractical Approach”, Oxford University Press, (2000). In someembodiments, glycopeptides can be synthesized using glycosylated aminoacids such that glycosylated amino acids, such as GlcNAc-Asn (V-Labs,Covington, La.), are incorporated at the appropriate glycosylation sitesof the peptide.

Vaccines of the present disclosure can be employed as an antiviral agentby administering the peptide topically, intranasally, or throughparenteral administration, such as through sub-cutaneous injection,intra-muscular injection, intravenous injection, intraperitonealinjection, or intra-dermal injection, to a warm-blooded animal, e.g.,humans, horses, other mammals, etc. The antiviral peptides can be usedindividually or in combination. Additionally, the antiviral peptide maybe administered alone or as part of a composition that further comprisesone or more pharmaceutically acceptable carriers, the proportion ofwhich is determined by the solubility and chemical nature of thepeptide, chosen route of administration and standard biologicaladministration. Because inventive peptides may target proteins on thesurfaces of the virus and/or the cell, to ensure efficacy, the carrierin such formulations should be free or substantially free (e.g., betterthan 90, 95, 98, or 99 wt %) of proteins that bind to the peptides.

Pharmaceutical Compositions

According to another aspect, the vaccines and deglycosylated proteins ofthe present disclosure can be included in a pharmaceutical ornutraceutical composition or formulation together with additional activeagents, carriers, vehicles, adjuvants, excipients, or auxiliary agentsidentifiable by a person skilled in the art upon reading of the presentdisclosure.

The vaccines of the present disclosure will advantageously comprise anadjuvant peptide in an effective adjuvant amount. As will be apparent toone skilled in the art, the optimal concentration of the adjuvantpeptide or peptides will necessarily depend upon the specific peptide(s)used, the characteristics of the patient, the immunogen used, and thenature of the viral infection for which the treatment or prophylaxis issought. These factors can be determined by those of skill in the medicaland pharmaceutical arts in view of the present disclosure. In general,the adjuvant peptides are most desirably administered at a concentrationlevel that will generally afford adjuvant activity without causing anyharmful or deleterious side effects. Generally, an effective adjuvantamount is desired. An effective adjuvant amount refers to an amount ofan adjuvant peptide which is capable of stimulating an immune responseto an administered immunogen.

Suitable adjuvants for inclusion in compositions of the presentdisclosure include those that are well known in the art, such ascomplete Freund's adjuvant (CFA) that is not used in humans, incompleteFreund's adjuvant (IFA), squalene, squalane, alum, and various oils, allof which are well known in the art, and are available commercially fromseveral sources, such as Novartis (e.g., MF59 adjuvant).

The pharmaceutical or nutraceutical compositions preferably comprise atleast one pharmaceutically acceptable carrier. In such pharmaceuticalcompositions, the vaccine or deglycosylated protein forms the “activecompound,” also referred to as the “active agent.” As used herein thelanguage “pharmaceutically acceptable carrier” includes solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions. A pharmaceutical composition isformulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g.,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol, or other syntheticsolvents; antibacterial agents such as benzylalcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediamine tetraacetic acid; buffers such as acetates.citrates, or phosphates and agents for the adjustment of tonicity suchas sodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes, ormultiple dose vials made of glass or plastic.

Suitable pharmaceutically acceptable carriers for the compositionscontaining the peptides are described in the standard pharmaceuticaltexts. See, e.g., “Remington's Pharmaceutical Sciences”, 18th Ed., MackPublishing Company, Easton, Pa. (1990). Specific non-limiting examplesof suitable pharmaceutically acceptable carriers include water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the composition can further contain minor amountsof auxiliary substances such as wetting or emulsifying agents, pHbuffering agents that enhance the antiviral effectiveness of thecomposition.

Subject as used herein refers to humans and non-human primates (e.g.guerilla, macaque, marmoset), livestock animals (e.g., sheep, cow,horse, donkey, and pig), companion animals (e.g., dog. cat), laboratorytest animals (e.g., mouse, rabbit. rat, guinea pig. hamster), captivewild animals (e.g., fox. deer), and any other organisms who can benefitfrom the agents of the present disclosure. There is no limitation on thetype of animal that could benefit from the presently described agents. Asubject regardless of whether it is a human or non-human organism may bereferred to as a patient, individual, animal, host, or recipient.

For parenteral administration, the peptides of the present disclosure orvaccines therefrom may be administered by intravenous, subcutaneous,intramuscular, intraperitoneal, or intradermal injection, alone or incompositions further comprising pharmaceutically accepted carriers. Foradministration by injection, it is preferred to use the antiviralpeptide in a solution in a sterile aqueous vehicle which may alsocontain other solutes such as buffers or preservatives as well assufficient quantities of pharmaceutically acceptable salts or of glucoseto make the solution isotonic. The antiviral peptides of the presentdisclosure can be obtained in the form of therapeutically acceptablesalts that are well-known in the art.

Pharmaceutical compositions suitable for an injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline. bacteriostatic water, CremophorEL™ (BASF, Parsippany. N.J.) or phosphate buffered saline (PBS). In allcases, the composition should be sterile and should be fluid to theextent that easy syringability exists. It should be stable under theconditions of manufacture and storage and be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing. for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof.

The proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Preventionof the action of microorganisms can be achieved by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as manitol, sorbitol, or sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecomposition in the required amount in an appropriate solvent with one ora combination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation include vacuumdrying and freeze-drying, which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Vaccines may also be administered orally. Oral compositions generallyinclude an inert diluent or an edible carrier. For the purpose of oraltherapeutic administration, the active compound can be incorporated withexcipients and used in the form of tablets, troches, or capsules, e.g.,gelatin capsules. Oral compositions can also be prepared using a fluidcarrier for use as a mouthwash. Pharmaceutically compatible bindingagents, or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

Because the peptides and vaccines of the present disclosure have shownactivity against respiratory viruses, they can also be delivered locallyto the respiratory system, for example to the nose, sinus cavities,sinus membranes or lungs. The peptide(s), vaccines, or pharmaceuticalcompositions containing one or more peptides or vaccines, can bedelivered to the respiratory system in any suitable manner, such as byinhalation via the mouth or intranasally. The present compositions canbe dispensed as a powdered or liquid nasal spray, suspension, nosedrops, a gel or ointment, through a tube or catheter, by syringe, bypacktail, by pledget, or by submucosal infusion. The peptides orvaccines may be conveniently delivered in the form of an aerosol sprayusing a pressurized pack or a nebulizer and a suitable propellant, e.g.,without limitation, dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be controlled by providing avalve to deliver a metered amount. Capsules and cartridges of, forexample, gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the peptide and a suitable powder base suchas lactose or starch. Examples of intranasal formulations and methods ofadministration can be found in PCT publications WO 01/41782, WO00/33813, and U.S. Pat. Nos. 6,180,603; 6,313,093; and 5,624,898. Thelatter-cited U.S. patents are incorporated herein by reference and forall purposes. A propellant for an aerosol formulation may includecompressed air, nitrogen, carbon dioxide, or a hydrocarbon based lowboiling solvent. The peptides or vaccines of the present disclosure canbe conveniently delivered in the form of an aerosol spray presentationfrom a nebulizer or the like. In some aspects, the active ingredientsare suitably micronised so as to permit inhalation of substantially allof the active ingredients into the lungs upon administration of the drypowder formulation, thus the active ingredients will have a particlesize of less than 100 microns, desirably less than 20 microns, andpreferably in the range 1 to 10 microns. In one embodiment, one or moreof the peptides or vaccines are packaged into a device that can delivera predetermined, and generally effective, amount of the peptide viainhalation, for example a nasal spray or inhaler.

Systemic administration can also be transmucosal or transdermal. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration may be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active compounds are formulated into ointments, salves, gels, orcreams as generally known in the art. The compounds can also be preparedin the form of suppositories (e.g., with conventional suppository basessuch as cocoa butter and other glycerides) or retention enemas forrectal delivery.

According to implementations, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to cell-specific antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811, which is incorporated by reference herein.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit faun as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

It will be appreciated by those skilled in the art that reference hereinto treatment extends to prophylaxis as well as the treatment ofestablished infections or symptoms. The peptides and vaccines of thepresent disclosure may be administered therapeutically orprophylactically. Treatment is preferably commenced before or at thetime of infection or at the time the mammal is exposed to a virus thatis capable of causing a viral respiratory infection, and continued untilvirus is no longer present or active in the respiratory tract. However,the treatment can also be commenced post-infection, after the mammal hasbeen exposed to a virus that is capable of causing a viral respiratoryinfection, or after the appearance of established symptoms of infection.

It will be further appreciated that the amount of an antiviral peptideof the present disclosure that is useful in treatment or prevention ofinfluenza will vary not only with the particular peptide selected butalso with the route of administration, the nature of the condition beingtreated, and the age and condition of the patient, and will ultimatelybe at the discretion of the attendant physician or veterinarian. Ingeneral however, a suitable dose will be in the range of from about 0.01to 750 mg/kg of body weight per day preferably in the range of 0.1 to100 mg/kg/day, most preferably in the range of 0.5 to 25 mg/kg/day.

The peptide or vaccine may be conveniently administered in unit dosageform, for example, containing 10 to 1500 mg, conveniently 20 to 1000 mg,most conveniently 50 to 700 mg of active ingredient per unit dosageform, e.g. 1 mg/kg equates to 75 mg/75 kg of body weight.

Preferably the concentration of immunogen for each strain of theinfluenza virus for inclusion in the vaccine is an amount which inducesan immune response without significant, adverse side effects. Suchamount will vary depending on which immunogen is used and the type andamount of adjuvant peptide included in the vaccine. Typically, a vaccinewill comprise immunogen in an amount of from about 1 to about 1000 μgper ml, more preferably from about 3 to about 300 μg per ml and mostpreferably about 10 μg to about 15 μg per ml, as measured by a SRDassay. Following an initial vaccination, subjects being vaccinated mayreceive one or several booster immunizations adequately spacedthereafter.

Toxicity and therapeutic efficacy of such compositions may be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected location to minimize potential damage to uninfectedcells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the disclosure, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of the activecompound (i.e., an effective dosage) may range from about 0.001 to 100g/kg body weight, or other ranges that would be apparent and understoodby artisans without undue experimentation. The skilled artisan willappreciate that certain factors can influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health or age of the subject, and other diseases present.

EXAMPLES

Without intent to limit the scope of the invention, exemplaryinstruments, apparatus, methods and their related results according tothe embodiments of the present invention are given below. Note thattitles or subtitles may be used in the examples for convenience of areader, which in no way should limit the scope of the invention.Moreover, certain theories are proposed and disclosed herein; however,in no way they, whether they are right or wrong, should limit the scopeof the invention so long as the invention is practiced according to theinvention without regard for any particular theory or scheme of action.

Example 1: Gene Construct for HA Expression

The Influenza H5N1 HA sequence was from the consensus H5, CHAS (Chen MW, et al. (2008) Proc Natl Acad Sci USA 105:13538-13543). The codons ofCHAS were optimized for expression by using human codons. The originalviral protease cleavage site PQRERRRKKRG (SEQ ID NO: 1) was mutated toPQRERG (SEQ ID NO: 2) in order to prevent proteins from the enzymaticcleavage to form HA I and HA2. The transmembrane region (residues:533-555) was replaced with the additional residuesLVPRGSPGSGYIPEAPRDGOAYVRKDGEWVLLSTFLGHHHHHH (SEQ ID NO: 3) at the Cterminus of the HA construct, where the thrombin cleavage site is initalics, the bacteriophage T4 fibritin foldon trimerization sequence isunderlined, and the His-tag is in bold (Stevens J. et al. (2006) Science312:404-410). The modified HA sequence was cloned into pTT vector forprotein expression (Durocher Y, et al. (2002) Nucleic Acids Res 30:E9).

Example 2: Protein Expression and Purification

The plasmid that encodes the secreted HA was transfected into the humanembryonic kidney cell lines of either HEK293EBNA (ATCC number CRL-10852)or the GnTI-HEK293S cells (Reeves P J, et al. (2002) Proc Natl Acad SciUSA 99:13419-13424) by using polyethyleneimine and was cultured inFreestyle 293 expression medium (Invitrogen, Carlsbad, Calif.)supplemented with O.S % bovine calf serum. The supernatant was collected72 h after transfection and cleared by centrifugation. HA proteins werepurified with Nickel-chelation chromatography as previously described(Wei C J, et al. (2008) J Virol 82:6200-6208) to obtain fullyglycosylated HA_(fg) and high-mannose-type HA_(hm). To obtain the HAprotein without sialylation—the desialylated HA_(ds)—the purifiedprotein was treated with 20 mM Clostridium neuraminidase (NA; Sigma) for2 h at 37° C. After the NA treatment, the protein was purified again tobe separated from the NA. The purified HA_(hm) was treated with Endo H(NEB) for 2 h at 37° C. to produce HA protein with a single GlcNAc atthe glycosylation sites, the monoglycosylated HA_(mg). All purified HAproteins were analyzed with SDS PAGE, glycan array and the massspectrometry (MS).

Example 3: Release of N-Glycans from Glycoproteins for MS Analysis

The purified HA glycoproteins were reduced with 10 mM dithiothreitol(DTT, Sigma) at 37° C. for 1 hour. Reduced sample was then alkylated by50 mM Iodoacetamide (IAA, Merck) in the dark for 1 hr and then wasdesalted by double distilled (ddH₂O) and dried in a speed vacuum. Thereduced and alkylated HA protein extracts were first digested withtrypsin (Roche) in an approximate ratio of enzyme to protein at 1:20(w/w) in 50 mM ammonium bicarbonate buffer pH 8.3 at 37° C. for 4 hrs,followed by secondary typsin (Roche) digestion, and then loaded on toreverse phase C18 Sep-Pak cartridge (Waters Corp). The Sample werefurthermore incubated with N-glycosidase F (Roche) in 50 mM ammoniumbicarbonate pH 8.3 at 37° C. for 16 hrs, and with two more N-glycosidaseF incubations. Released N-glycans were separated frompeptides/glycopeptides by C 18 Sep-Pak cartridge procedure withN-glycans collected in 5% acetic acid (AcOH), the flow-through fraction.The peptides were eluted in 20%, 40% and 60% 1-propanol with 5% AcOH.

Example 4: MALDI-MS and MS/MS Analysis

All glycan samples were permethylated using the NaOH/dimethyl sulfoxideslurry method. The NaOH/DMSO slurry was mixed with dried glycan samplesin screw-capped glass tube and 300 μL iodmethane (Merck) was added intotube, and the tube was gently vortexed for 25 min. Reaction wasterminated by adding ˜1 ml ddH2O drop-wise, and then an equal volume ofchloroform was added. Permethylated glycans were extracted into thebottom organic layer and additional NaOH as well as other hydrophiliccontaminants were removed by repeated extraction with ddH₂O. Chloroformwas evaporated by nitrogen gas. For glycan profiling, permethylatedglycans in acetonitrile were mixed 1:1 with 10 mg/ml of2,5-dihydroxybenzoic acid (DHB) in 50% acetonitrile, spotted on heatedtarget plate, and recrystallized on-plate with acetonitrile. Dataacquisition was performed on ABI 4700Proteomics Analyzer (AppliedBiosystems) operated in the reflectron mode. Laser shots (5 Hz; 10 shotsper spectrum) were accumulated until a satisfactory signal to noiseratio was achieved when combined and smoothed. On the TOF/TOFinstrument, high-energy CID MS/MS data were manually acquired andtypically comprised a total of 40 sub-spectra of 125 laser shots at alaser energy setting of 5000-5500.

Example 5: Glycan Microarray Fabrication

Twenty-four sialic acid-containing glycans designed for HAs wereprepared chemically and used for array fabrication. Microarrays wereprinted (BioDot; Cartesian Technologies) by robotic pin (SMP3; TeleChemInternational) deposition of ˜0.7 nL of various concentrations ofamine-containing glycans in printing buffer (300 mM phosphate buffer, pH8.5, containing 0.005% Tween 20) from a 384-well plate onto NHS-coatedglass slides (Nexterion H slide; SCHOTT North America). The slides forsialosides were spotted with solutions of glycans 1-17 and 21-27 withconcentrations of 100 μM in each row for one glycan from bottom to top,with 12 replicates horizontally placed in each subarray, and each slidewas designed for 16 grids for further incubation experiments. Printedslides were allowed to react in an atmosphere of 80% humidity for anhour followed by desiccation overnight, and they were stored at roomtemperature in a desiccator until use. Before the binding assay, theseslides were blocked with ethanolamine (50 mM ethanolamine in boratebuffer, pH 9.2) and then washed with water and PBS buffer, pH 7.4,twice.

Example 6: Hemagglutinin Labeling with Cy3-NHS Ester

Each HA protein sample was diluted with PBS (pH=7.4) to the finalconcentration of I mg/mL, and then labeled with Cy3Mono NHS Ester (5 μL,0.2 mg/ml) (GE Healthcare, UK). After the reactions had proceeded 18 hron ice, 20 μL of 500 mM of glycine in PBS was added to each tube toquench the reactions. Then the solutions were incubated on ice for anadditional 30 min Non-reactive dye molecules were removed by passingeach solution through a size exclusion spin filter (Microcon YM-30,Millipore, USA) with a molecular weight cutoff of 30 kDa. In order toobtain the ratio of dye/protein, each sample of labeled protein wasdiluted with PBS for the dual absorbance measurements at 280 nm (forprotein) and at 552 nm (for Cy3; the molar extinction coefficient is150,000 M⁻¹cm⁻¹ at this wavelength) by using NanoDrop ND-IOOSpectrophotometer (NanoDrop Technologies, USA). After correcting thecalculation for the absorbance of CyDye at 280 nm (approximately 8% ofthe absorbance at 552 nm), the ratios of dye/protein were generated fromthe results of dual-absorbance measurements.

Example 7: Indirect Binding Assay

HA glycosylated variants were prepared in 0.005% Tween 20/PBS buffer, pH7.4, and added to cover the grid on glycan array with application of acoverslip. After incubation in a humidified chamber with shaking for 1h, the slides were washed three times with 0.005% Tween 20/PBS buffer,pH 7.4. Next, rabbit anti-H5N1 HA antibody was added to the slides andincubated in a humidified chamber for 1 h. After washing the slides with0.005% Tween 20/PBS buffer three times, Cy3-conjugated goat anti-rabbitIgG antibody was added to the slides and incubated in a humidifiedchamber for another 1 h. The slides were washed three times with 0.05%Tween 20/PBS buffer, pH 7.4; three times with PBS buffer, pH 7.4; andthree times with H2O, and then dried. The slides were scanned at 595 nm(for Cy3) with a microarray fluorescence chip reader (GenePix Pro 6.0;Molecular Devices).

Example 8: Direct Binding Assay

Cy3-labeled HA proteins with different glycosylation were prepared in0.005% Tween 20IPBS buffer (pH 7.4) and added to cover the grid onglycan array with application of a coverslip. After incubation in ahumidifying chamber with shaking for 1 h, the slides were washed threetimes with 0.005% Tween 20/PBS buffer (pH 7.4), three times with PBSbuffer (pH 7.4), and three times with H2O and dried. The slide wasscanned at 595 nm (for Cy3) with a microarray fluorescence chip reader(GenePix Pro 6.0, Molecular Devices, USA).

Example 9: Microneutralization Assay

The freshly prepared H5N1 (NIBRG-14) virus (National Institute forBiological Standards and Control, Potters Bar, U.K.) was quantified withthe median tissue culture infectious dose (TCID₅₀). The 100-fold TCID₅₀of virus was mixed in equal volume with 2-fold serial dilutions of serumstock solution in 96-well plates and incubated for 1 h at 37° C. Themixture was added onto the MDCK cells (1.5×10⁴ cells per well) on theplates, followed by incubation at 37° C. for 16-20 h. The cells werewashed with PBS, fixed in acetone/methanol solution (vol/vol 1:1), andblocked with 5% skim milk. The viral antigen was detected by indirectELISA with a mAb against influenza A NP (Sui J H, et al. (2009) NatStruct Mol Biol 16:265-273).

Example 10: Mice, Vaccination, and Challenge

Female 6- to 8-week-old BALB/c mice (n=15) were immunizedintramuscularly with 20 μg of purified HA_(fg) or HA_(mg) proteins in 50μL of PBS, pH 7.4, and mixed with 50 μL of 1 mg/mL aluminum hydroxide(Alum; Sigma) at weeks 0 and 2. Blood was collected 14 days afterimmunization, and serum samples were collected from each mouse. Theimmunized mice were challenged intranasally with a genetically modifiedH5N1 virus, NIBRG-14, with a lethal dose (100-fold lethal dose to 50% ofmice). The mice were monitored daily for 14 days after the challenge forsurvival. All animal experiments were evaluated and approved by theInstitutional Animal Care and Use Committee of Academia Sinica.

Example 11: Hemagglutination (HA) Assay

Hemagglutination of chicken red blood cells (cRBCs, Lampire BiologicalLaboratories, Pipersville, Pa.) is carried out in round bottom 96-wellmicrotiter plates by preparing two-fold dilutions of viral samples inPBS, as described in Jones, et al., Journal of Virology,80(24):11960-11967 (2006). Titer is reported as hemagglutinating unitsper 50 μL (HAU/50 μL) of sample.

Example 12: Purification of Viral Hemagglutinin

Viron-associated hemagglutinin (HA) was purified from influenza virusparticles as described in Johansson, et al., Journal of Virology, 1989,Vol. 63(3), p. 1239-1246, with modifications. Briefly, virus iscollected from the allantoic fluid of infected hen's eggs and sucrosepurified as described above. Pellets are resuspended in 0.5 mL of sodiumacetate buffer (0.05 M sodium acetate, 2 mM CaCl.sub.2, 0.2 mM EDTA, pHto 7.0), homogenized through an 18-gauge needle, and mixed with an equalvolume of 15% octylglucoside (octyl-B-d-thioglucoside; FisherScientific, Norcross, Ga.) in sodium acetate buffer, followed byvigorous vortexing for 5 minutes. This suspension is centrifuged at18,400×g for 60 minutes at 4° C., and the supernatant carefully removedand reserved as the HA-rich fraction. Two percent aqueous cetyltrimethyl-ammonium bromide (CTAB, Bio-World, Dublin, Ohio) is added tothe HA fraction to a final concentration of 0.1% CTAB, and the sample isapplied to a DEAE-Sephadex (A-50; GE Healthcare, Uppsala, Sweden)ion-exchange column (bed, 0.7 cm×6.0 cm) previously swollen andequilibrated with 0.05 M Tris-hydrochloride (pH 7.5) containing 0.1%octylglucoside. Twenty 0.5 mL fractions were collected by gravity withlow salt HA elution buffer (0.05 M TrisHCl, 0.1 M NaCl, 0.1% TritonX-100, pH to 7.5) and again with a high salt HA elution buffer (0.05 MTrisHCl, 0.2 M NaCl, 0.1% Triton X-100, pH to 7.5). Individual fractionsare assayed for HA activity and analyzed for purity bySDS-polyacrylamide gel electrophoresis under non-reducing conditionsfollowed by staining with colloidal Commassie. Protein concentration isdetermined by BCA assay as per manufacturer's instructions (Pierce,Rockford, Ill.).

Example 13: Array Data Analysis

The software GenePix Pro (Axon Instruments) was used for thefluorescence analysis of the extracted data. The local background wassubtracted from the signal at each spot. The spots with obvious defects,no detectable signal, or a net fluorescence of <100 were removed fromthe analysis. The “medians of ratios” from replicate spots were averagedin the same array. The profiling of HA binding to the array (FIG. 2A)and the determination of association constant (FIG. 2B) were performedunder the same conditions on the same array to ensure the data werenormalized.

To determine the K_(D,surf) value, the equilibrium binding data wereanalyzed by fitting the data to the Langmuir isotherms (equation 1),assuming that ligands bound to one or two independent sites, using thecommercial nonlinear regression program GradPad PRISM (GraphPad).

F_(max) is the maximum fluorescence intensity, a measure of the amountof active carbohydrate on the surface; (P) is the total HA proteinsconcentration, and K_(D,surf) is the equilibrium dissociation constantbetween the surface carbohydrates and the proteins.

The K_(D,surf) values of each sample were repeated and calculated atleast 4 times to derive mean of K_(D,surf). By using K_(D,surf) values,the thermodynamic parameters can be derived from the equations (2) and(3).F _(obs) =F _(max)(P)/(K _(D,surf)+(P))  {Equation 1}K _(D,surf) =K _(A,surf) ⁻¹  {Equation 2}ΔG _(mum) =RT ln(K _(A,surf))  {Equation 3}

K_(A,surf) represents the association constants in equation (1). Inequation (2), R=1.987 cal mol⁻¹ K⁻¹; T is the absolute temperature, andthe experiments were performed at 298 K. These values of each samplewere calculated by using Microsoft Excel. The statistical analysis ofK_(D,surf) in different HA glycoforms was performed with one-way ANOVAby using GraphPad PRISM (GraphPad).

Example 14: Determination of HA-Specific Antibodies in Serum by ELISA

The HA proteins were purified from HEK293 and coated on the 96-wellplates (5 μg/mL) overnight. The mouse serum was diluted 100-fold to bethe stock serum for the measurement of HA binding. The HA-coated plateswere incubated with serum in 2-fold serial dilutions for 1 h.HA-specific IgG was detected by using HRP-conjugated anti-mouseantibodies. The endpoint dilution was calculated by picking the dilutionfor which the readout was above that of the 1:50 dilution of preimmuneserum (Stevens J. et al. (2006) Science 312:404-410). Antiserum fromrabbit was prepared by LTK BioLaboratories. Rabbit was immunized byabout 0.25-0.35 mg of HA proteins mixed with complete or incompleteadjuvants. Blood was collected from rabbit after six immunizations, witha schedule of one immunization every 2 weeks.

Example 15: Glycosylation Site Analysis for HA

A total of 297 full-length HA sequences from H1, H3, and H5 influenzaviruses were retrieved from the National Center for BiotechnologyInformation database and aligned by ClustalW2 program in EMBL-EBI(Larkin M A, et al. (2007) Bioinformatics 23:2947-2948). The sequencesdate from the years 1918 to 2000s and were isolated from humans. Toreduce redundancy, strains in one country were only selected one timefor analysis. Sequences used for alignment include: H1 (AAX56530,AAY78939, ABA18037, ABB51962, ABC42750, ABD60867, ABD62061, ABE11690,ABF47869, ABF82830, ABF82852, ABG37362, ABI19015, ABI21211, ABI95294,ABI96103, ABK39995, ABK57092, ABK57093, ABK79970, AB032948, AB032970,ABR15885, ABS71664, and ABS76427); H3 (AAT08000, AAT12654, AAX11455,AAY58320, AAZ32943, AAZ43394, ABA26700, ABA26777, ABB51961, ABB71825,ABC42596, ABC42607, ABC42629, ABC43017, ABD15713, ABD59850, ABD61359,ABF17954, ABG37450, and ABG37461); H5 (AAS65618, AAT39065, AAT73273,AAT73274, AAT73275, AAT84153, AAV32636, ABC72655, ABD28180, ABD28182,ABE97624, ABI16504, ABI36144, ABI36439, AB010181, AB036644, andABP51968). N-linked glycosylations of HA sequences were predicted bycenter of biological sequence analysis prediction severs(www.cbs.dtu.dk/services/). For glycosylation of asparagine (Asn), thesequences contain amino acid pattern Asn-X_(aa)-(Ser/Thr), where Xaa canbe any amino acid except for proline (Gavel Y, et al. (1990) Protein Eng3:433-442), followed by serine or threonine. The results for dataanalysis were prepared by using the PRISM program (GraphPad) and Jalview(Waterhouse A M, et al. (2009) Bioinformatics 25:1189-1191).

Example 16: Chemical Method for Synthesis of HA Glycopeptides

The stepwise synthesis of HA glycopepides can be carried out by thedimethylphosphinothioic mixed anhydride (Mpt-MA) method (Inazu, T., etal. (1997) in Peptide Chemistry 1996 (Kitada, C. ed.) pp. 41-44, ProteinResearch Foundation, Osaka). HA glycoproteins can be synthesized by athioester method with a consensus sequence “Asn-X-Ser/Thr” forN-glycosylation but no sugar chains. The peptide fragment is prepared byan automatic synthesizer using a supplied Boc-strategy program. Thecoupling reaction is performed by using DCC/HOBt as the activatingreagent. Asn(GlcNAc) residue is coupled by a Mpt-MA method usingBoc-Asn(GlcNAc)-OH (3 equiv). The coupling reactions are performed for 1h and repeated with monitoring. After treatment with anhydrous HFcontaining 10% anisole and HPLC purification, a glycopeptide thioesteris obtained. This glycopeptide thioester segment is coupled with theother peptide segment, which was prepared separately by the thioestersegment condensation method. After deprotections and disulfide bondformation, the GlcNAc-HA analog is obtained.

Alternately, a convergent method synthesis of HA glycopepides can becarried out by the coupling reaction of the β-carboxyl group of peptidylAsn with glycosylamine. (See Cohen-Abisfeld, S. T., and Lansbury, P. T.(1993) J. Am. Chem. Soc. 115, 10531-10537)

Example 17: Chemo-Enzymatic Method for Synthesis of HA Glycopeptides

The preparation of a glycopeptide containing a complex oligosaccharidecan be performed by using enzymatic methods in conjunction with chemicalmethods. Synthesis of N-glycopeptides can be performed using thetransglycosylation activity of endo-β-N-acetylglucosaminidase(endo-β-GlcNAc-ase). (Takegawa, K., et al. (1995) J. Biol. Chem. 270,3094-3099). Endo-β-GlcNAc-ase hydrolyzes the glycosidic bond between theN,N′-diacetylchitobiose moiety of a N-linked oligosaccharide, andtransfers the released oligosaccharide fragment to a hydroxyl compound.The synthesis of N-glycopeptides using endo-β-GlcNAc-ase can beperformed in two steps. First, a GlcNAc-containing peptide is preparedby a chemical route. Then an oligosaccharide fragment of a glycosyldonor is transferred to the GlcNAc moiety of the glycopeptide as aglycosyl acceptor by the transglycosylation reaction ofendo-β-GlcNAc-ase.

Although specific embodiments of the invention have been describedherein for purposes of illustration, various modifications may be madewithout deviating from the spirit and scope of the invention.Accordingly, the invention is not limited except as by the appendedclaims.

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

The invention claimed is:
 1. An immunogenic composition for raising animmune response to a pathogen of viral, bacterial, fungal or otherorigin, the composition comprising: an antigen glycoprotein from thepathogen of viral, bacterial, fungal or other origin, wherein theglycoprotein comprises at least one glycosylation site, wherein theantigen glycoprotein is partially glycosylated at one or moreglycosylation sites, and wherein the partially glycosylated antigenglycoprotein comprises a partial glycosylation site to which a glycanthat consists of one, two, or three sugar residues is bound.
 2. Theimmunogenic composition according to claim 1, in which the virus isselected from influenza virus, respiratory syncytial virus (RSV),chlamydia, adenovirdiae, mastadenovirus, aviadenovirus, herpesviridae,herpes simplex virus 1, herpes simplex virus 2, herpes simplex virus 5,herpes simplex virus 6, leviviridae, levivirus, enterobacteria phaseMS2, allolevirus, poxviridae, chordopoxvirinae, parapoxvirus,avipoxvirus, capripoxvirus, leporiipoxvirus, suipoxvirus,molluscipoxvirus, entomopoxvirinae, papovaviridae, polyomavirus,papillomavirus, paramyxoviridae, paramyxovirus, parainfluenza virus 1,mobillivirus, measles virus, rubulavirus, mumps virus, pneumonovirinae,pneumovirus, metapneumovirus, avian pneumovirus, human metapneumovirus,picornaviridae, enterovirus, rhinovirus, hepatovirus, human hepatitis Avirus, cardiovirus, andapthovirus, reoviridae, orthoreovirus, orbivirus,rotavirus, cypovirus, fijivirus, phytoreovirus, oryzavirus,retroviridae, mammalian type B retroviruses, mammalian type Cretroviruses, avian type C retroviruses, type D retrovirus group,BLV-HTLV retroviruses, lentivirus, human immunodeficiency virus 1, humanimmunodeficiency virus 2, spumavirus, flaviviridae, hepatitis C virus,flavivirus, Dengue virus, hepadnaviridae, hepatitis B virus,togaviridae, alphavirus sindbis virus, rubivirus, rubella virus,rhabdoviridae, vesiculovirus, lyssavirus, ephemerovirus,cytorhabdovirus, necleorhabdovirus, arenaviridae, arenavirus,lymphocytic choriomeningitis virus, Ippy virus, lassa virus,coronaviridae, coronavirus, and torovirus.
 3. The immunogeniccomposition according to claim 2, in which the viral peptide, protein,polypeptide, or a fragment thereof is a surface glycoprotein selectedfrom influenza virus neuraminidase, influenza virus hemagglutinin,influenza virus M2 protein, human respiratory syncytial virus(RSV)-viral proteins, RSV F glycoprotein, RSV G glycoprotein, herpessimplex virus (HSV) viral proteins, herpes simplex virus glycoproteinsgB, gC, gD, and gE, Chlamydia MOMP and PorB antigens, core protein,matrix protein or other protein of Dengue virus, measles virushemagglutinin, herpes simplex virus type 2 glycoprotein gB, poliovirus IVP1, envelope glycoproteins of HIV 1, hepatitis B surface antigen,diptheria toxin, Streptococcus 24M epitope, Gonococcal pilin,pseudorabies virus g50 (gpD), pseudorabies virus II (gpB), pseudorabiesvirus III (gpC), pseudorabies virus glycoprotein H, pseudorabies virusglycoprotein E, transmissible gastroenteritis glycoprotein 195,transmissible gastroenteritis matrix protein, swine rotavirusglycoprotein 38, swine parvovirus capsid protein,Serpulinahydodysenteriae protective antigen, bovine viral diarrheaglycoprotein 55, Newcastle disease virus hemagglutinin-neuraminidase,swine flu hemagglutinin, swine flu neuraminidase, foot and mouth diseasevirus, hog cholera virus, swine influenza virus, African swine fevervirus, Mycoplasma liyopneutiioniae, infectious bovine rhinotracheitisvirus, infectious bovine rhinotracheitis virus glycoprotein E,glycoprotein G, infectious laryngotracheitis virus, infectiouslaryngotracheitis virus glycoprotein G or glycoprotein I, a glycoproteinof La Crosse virus, neonatal calf diarrhea virus, Venezuelan equineencephalomyelitis virus, punta toro virus, murine leukemia virus, mousemammary tumor virus, hepatitis B virus core protein and hepatitis Bvirus surface antigen or a fragment or derivative thereof, antigen ofequine influenza virus or equine herpes virus, including equineinfluenza virus type A/Alaska 91 neuraminidase, equine influenza virustypeA/Miami 63 neuraminidase, equine influenza virus type A/Kentucky 81neuraminidase equine herpes virus type 1 glycoprotein B, and equineherpes virus type 1 glycoprotein D, antigen of bovine respiratorysyncytial virus or bovine parainfluenza virus, bovine respiratorysyncytial virus attachment protein (BRSV G), bovine respiratorysyncytial virus fusion protein (BRSV F), bovine respiratory syncytialvirus nucleocapsid protein (BRSVN), bovine parainfluenza virus type 3fusion protein, bovine parainfluenza virus type 3 hemagglutininneuraminidase, bovine viral diarrhea virus glycoprotein 48 andglycoprotein 53, glycoprotein E of Dengue virus, and glycoprotein E1 orE2 of human hepatitis C virus.
 4. The immunogenic composition accordingto claim 1, wherein the partial glycosylation site is located at anO-glycosylation or N-glycosylation site.
 5. The immunogenic compositionaccording to claim 1, wherein the partial glycosylation site is anN-glycosylation site comprising an amino acid sequence ofasparagine-X_(aa)-serine and asparagine-X_(aa)-threonine, where X_(aa)is any amino acid except proline.
 6. The immunogenic compositionaccording to claim 1, wherein the partially glycosylated viral antigenis a mono-, di-, or tri-glycosylated influenza virus hemagglutinin (HA),neuraminidase (NA) or M2 protein, or immunogenic fragments thereof. 7.The immunogenic composition according to claim 6, wherein the partiallyglycosylated viral antigen is an influenza virus hemagglutininglycosylated with N-acetylglucosamine (GlcNAc) and/or mannose.
 8. Theimmunogenic composition according to claim 1, wherein the virus isrespiratory syncytial virus (RSV), and the partially glycosylated viralantigen is a mono-, di-, or triglycosylated RSV F (fusion), G(attachment) of SH (small hydrophobic) glycoprotein, or immunogenicfragments thereof.
 9. The immunogenic composition according to claim 8,wherein the mono-glycosylated RSV G protein sequence (SEQ ID NO: 12)comprises a partial glycosylation site at an asparagine residue at oneor more N-glycosylation sites.
 10. The immunogenic composition accordingto claim 1, wherein the virus is a flavivirus, and the partiallyglycosylated viral antigen is a mono-, di-, or tri-glycosylated Denguevirus envelope glycoprotein M, glycoprotein E, or immunogenic fragmentsthereof.
 11. The immunogenic composition according to claim 10, whereinthe mono-glycosylated Dengue virus envelope glycoprotein E (SEQ ID NO:13) comprises the partial glycosylation site to which the glycan thatconsists of one sugar residue is bound at an asparagine residue at oneor more N-glycosylation sites selected from N67 and N153.
 12. Theimmunogenic composition according to claim 1, wherein the virus is ahepatitis C virus, and the partially glycosylated viral antigen is amono-, di-, or tri-glycosylated hepatitis C envelope glycoprotein E1,glycoprotein E2, or immunogenic fragments thereof.
 13. The immunogeniccomposition according to claim 12 wherein the mono-glycosylatedhepatitis C envelope glycoprotein E1 (SEQ ID NO: 14) comprises a partialglycosylation site at an asparagine residue at one or moreN-glycosylation sites selected from N196, N209, N234, N305, and N325.14. The immunogenic composition according to claim 1, wherein the virusis a human immunodeficiency virus (HIV), and the partially glycosylatedviral antigen is a mono-, di-, or triglycosylated HIV envelopeglycoprotein gp120, transmembrane glycoprotein gp41, or immunogenicfragments thereof.
 15. The immunogenic composition according to claim 14wherein the mono-glycosylated HIV envelope glycoprotein gp120 (SEQ IDNO: 15) comprises a partial glycosylation site at an asparagine residueat one or more N glycosylation sites.
 16. A vaccine comprising: (a) animmunogenic polypeptide comprising a viral surface glycoprotein, orimmunologically active fragments thereof that comprises a partialglycosylation site to which a glycan that consists of one, two, or threesugar residues is bound; and (b) optionally, an adjuvant, wherein thevaccine is capable of eliciting an immune response against a virus. 17.The vaccine according to claim 16, wherein the virus is an influenzavirus and the viral glycoprotein is hemagglutinin (HA), neuraminidase(NA) or M2.
 18. The vaccine according to claim 17, wherein the influenzavirus is selected from the group consisting of an avian influenza virus,an animal influenza virus and a human influenza virus.
 19. A method forimmunizing a mammal against a viral infection, the method comprising:administering to the mammal susceptible to infection by the respiratoryvirus the vaccine of claim 16.